IL-1 eta DNA and polypeptides

ABSTRACT

The invention is directed to novel, purified and isolated IL-1 eta polypeptides and fragments thereof, the polynucleotides encoding such polypeptides, processes for production of recombinant forms of such polypeptides, antibodies generated against these polypeptides, peptides derived from these polypeptides, and uses thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation in part of U.S. applicationsSerial Nos. 60/135,758, 60/162,331, and PCT/US00/14435, filed May 25,1999, Oct. 29, 1999, and May 25, 2000, respectively. The entiredisclosures of these applications are relied upon and incorporated byreference herein.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The invention is directed to novel, purified and isolated IL-1eta polypeptides and fragments thereof, the polynucleotides encodingsuch polypeptides, processes for production of recombinant forms of suchpolypeptides, antibodies generated against these polypeptides,fragmented peptides derived from these polypeptides, and uses thereof.

[0004] 2. Description of Related Art

[0005] Interleukin-1 (IL-1) is a member of a large group of cytokineswhose primary function is to mediate immune and inflammatory responses.There are seven known IL-1 family members which include IL-1 alpha(IL-1α), IL-1 beta (IL-1β), IL-1 receptor antagonist (IL-1ra), IL-1delta (IL-1δ), IL-1 epsilon (IL-1ε), IL-1 zeta (IL-1ξ) and IL-18(previously known as IGIF and sometimes IL-1 gamma). IL-1 that issecreted by macrophages is actually a mixture of mostly IL-1β and someIL-1α (Abbas et al., 1994). IL-1α and IL-1β, which are first produced as33 kD precursors that lack a signal sequence, are further processed byproteolytic cleavage to produce secreted active forms, each about 17 kD.Additionally, the 33 kD precursor of IL-1α is also active. Both forms ofIL-1 are the products of two different genes located on chromosome 2.Although the two forms are less than 30 percent homologous to eachother, they both bind to the same receptors and have similar activities.

[0006] IL-1ra, a biologically inactive form of IL-1, is structurallyhomologous to IL-1 and binds to the same receptors. Additionally, IL-1rais produced with a signal sequence which allows for efficient secretioninto the extracellular region where it competitively competes with IL-1(Abbas et al., 1994).

[0007] The IL-1 family of ligands binds to a family of two IL-1receptors, which are members of the Ig superfamily. IL-1 receptorsinclude the 80 kDa type I receptor (IL-1RI) and a 68 kDa type IIreceptor (IL-1RII). IL-1 ligands can also bind to a soluble proteolyticfragment of IL-1RII (sIL-1RII) (Colotta et al., 1993).

[0008] The major source of IL-1 is the activated macrophage ormononuclear phagocyte. Other cells that produce IL-1 include epithelialand endothelial cells (Abbas et al., 1994). IL-1 secretion frommacrophages occurs after the macrophage encounters and ingestsgram-negative bacteria. Such bacteria contain lipopolysaccharide (LPS)molecules, also known as endotoxin, in the bacterial cell wall. LPSmolecules are the active components that stimulate macrophages toproduce tumor necrosis factor (TNF) and IL-1. In this case, IL-1 isproduced in response to LPS and TNF production. At low concentrations,LPS stimulates macrophages and activates B-cells and other hostresponses needed to eliminate the bacterial infection; however, at highconcentrations, LPS can cause severe tissue damage, shock, and evendeath.

[0009] The biological functions of IL-1 include activating vascularendothelial cells and lymphocytes, local tissue destruction, and fever(Janeway et al., 1996). At low levels, IL-1 stimulates macrophages andvascular endothelial cells to produce IL-6, upregulates molecules on thesurface of vascular endothelial cells to increase leukocyte adhesion,and indirectly activates inflammatory leukocytes by stimulatingmononuclear phagocytes and other cells to produce certain chemokinesthat activate inflammatory leukocytes. Additionally, IL-1 is involved inother inflammatory responses such as induction of prostaglandins, nitricoxide synthetase, and metalloproteinases. These IL-1 functions arecrucial during low level microbial infections. However, if the microbialinfection escalates, IL-1 acts systemically by inducing fever,stimulating mononuclear phagocytes to produce IL-1 and IL-6, increasingthe production of serum proteins from hepatocytes, and activating thecoagulation system. Additionally, IL-1 does not cause hemorrhagicnecrosis of tumors, suppress bone marrow stem cell division, and IL-1 islethal to humans at high concentrations.

[0010] Given the important function of IL-1, there is a need in the artfor additional members of the IL-1 ligand and IL-1 receptor families. Inaddition, in view of the continuing interest in protein research and theimmune system, the discovery, identification, and roles of new proteins(such as the human IL-1 eta of the invention) and their inhibitors, areat the forefront of modern molecular biology and biochemistry. Despitethe growing body of knowledge, there is still a need in the art for theidentity and function of proteins involved in cellular and immuneresponses.

SUMMARY OF THE INVENTION

[0011] The invention aids in fulfilling these various needs in the artby providing isolated polynucleotides and polypeptides encoded by thepolynucleotides for the novel IL-1 family ligand termed “IL-1 eta.”Thus, in one aspect, the invention is directed to isolated novelpolynucleotide molecules of IL-1 eta comprising the nucleotide residues112-585 of SEQ ID NO:1 and to the isolated polynucleotide moleculesencoding the amino acid sequence of SEQ ID NO:2, as well aspolynucleotide molecules complementary to these sequences.

[0012] Both single-stranded and double-stranded RNA and DNA moleculesare encompassed by the invention, as well as polynucleotide moleculesthat hybridize to a denatured, double-stranded DNA comprising all or aportion of SEQ ID NO:1 and/or a DNA that encodes the amino acidsequences set forth in SEQ ID NO:2. Also encompassed are isolatedpolynucleotide molecules that are derived by in vitro mutagenesis ofpolynucleotide molecules comprising the coding region of SEQ ID NO:1,that are degenerate from polynucleotide molecules comprising thesequence of SEQ ID NO:1, and that are allelic variants of DNA of theinvention. The invention also encompasses recombinant vectors thatdirect the expression of these polynucleotide molecules and host cellstransformed or transfected with these vectors.

[0013] In addition, the invention encompasses methods of using the DNAnoted above to identify DNA encoding proteins having activitiesassociated with IL-1 family ligands and receptors.

[0014] In addition, these polynucleotides can be used to identify thehuman chromosomes with which the polynucleotides are associated. Thus,since the IL-1 eta polynucleotides map to chromosome 2, DNA encodingIL-1 eta polypeptides may be used to identify human chromosome 2.Accordingly, these polynucleotides may also be used to map genes onhuman chromosome 2; to identify genes associated with certain diseases,syndromes, or other human conditions associated with human chromosome 2;and to study cell signal transduction and the immune system.

[0015] The invention also encompasses the use of sense or antisenseoligonucleotides from the polynucleotides of SEQ ID NO:1 to inhibit theexpression of the respective polynucleotide encoded by the genes of theinvention.

[0016] The invention also encompasses isolated polypeptides andfragments of IL-1 eta as encoded by these polynucleotide molecules,including soluble polypeptide portions of SEQ ID NO:2. The inventionfurther encompasses methods for the production of these polypeptides,including culturing a host cell under conditions promoting expressionand recovering the polypeptide from the culture medium. Especially, theexpression of these polypeptides in bacteria, yeast, plant, insect, andanimal cells is encompassed by the invention.

[0017] In general, the polypeptides of the invention can be used tostudy cellular processes such as immune regulation, cell proliferation,cell death, cell migration, cell-to-cell interaction, and inflammatoryresponses. In addition, these polypeptides can be used to identifyproteins associated with IL-1 eta ligands.

[0018] In addition, the invention includes assays utilizing thesepolypeptides to screen for potential inhibitors of activity associatedwith polypeptide counter-structure molecules, and methods of using thesepolypeptides as therapeutic agents for the treatment of diseasesmediated by polypeptide counter-structure molecules. Further, methods ofusing these polypeptides in the design of inhibitors (e.g., engineeredreceptors that act as inhibitors) thereof are also an aspect of theinvention.

[0019] Further encompassed by this invention is the use of the IL-1 etapolynucleotide sequences, predicted amino acid sequences of thepolypeptide or fragments thereof, or a combination of the predictedamino acid sequences of the polypeptide and fragments thereof for use insearching an electronic database to aid in the identification of samplepolynucleotides and/or proteins.

[0020] Isolated polyclonal or monoclonal antibodies that bind to thesepolypeptides are also encompassed by the invention, in addition the useof these antibodies to aid in purifying the polypeptides of theinvention.

BRIEF DESCRIPTION OF THE FIGURES

[0021]FIG. 1 presents the nucleotide sequence of IL-1 eta (SEQ ID NO:1).

[0022]FIG. 2 presents the amino acid sequence of IL-1 eta (SEQ ID NO:2).

[0023]FIG. 3 is a table summarizing expression data of IL-1 eta. “-”indicates that the mRNA was looked for but not found; a blank spaceindicates that the analysis was not done for that particular gene/RNAcombination.

DETAILED DESCRIPTION OF THE INVENTION

[0024] The polynucleotide molecules encompassed in the invention includethe following nucleotide sequence: Name: IL-1 eta 1 GGCACGAGGTTCCTCCCCAC TCTGTCTTTC TCACCTCTCC TTCACTTTTC (SEQ ID NO:1) 51 CTAGCCTCCTCACCACCATC TGATCTATCT TGTTCTCTTC ACAAAAGGCT 101 CTGAAGACAT CATGAACCCACAACGGGAGG CAGCACCCAA ATCCTATGCT 151 ATTCGTGATT CTCGACAGAT GGTGTGGGTCCTGAGTGGAA ATTCTTTAAT 201 AGCAGCTCCT CTTAGCCGCA GCATTAAGCC TGTCACTCTTCATTTAATAG 251 CCTGTAGAGA CACAGAATTC AGTGACAAGG AAAAGGGTAA TATGGTTTAC301 CTGGGAATCA AGGGAAAAGA TCTCTGTCTC TTCTGTGCAG AAATTCAGGG 351CAAGCCTACT TTGCAGCTTA AGGAAAAAAA TATCATGGAC CTGTATGTGG 401 AGAAGAAAGCACAGAAGCCC TTTCTCTTTT TCCACAATAA AGAAGGCTCC 451 ACTTCTGTCT TTCAGTCAGTCTCTTACCCT GGCTGGTTCA TAGCCACCTC 501 CACCACATCA GGACAGCCCA TCTTTCTCACCAAGGAGAGA GGCATAACTA 551 ATAACACTAA CTTCTACTTA GATTCTGTGG AATA

[0025] The amino acid sequence of the polypeptides encoded by thenucleotide sequence of the invention include: Name: IL-1 eta(polypeptide) 1 MNPQREAAPK SYAIRDSRQM VWVLSGNSLI AAPLSRSIKP VTLHLIACRD(SEQ ID NO:2) 51 TEFSDKEKGN MVYLGIKGKD LCLFCAEIQG KPTLQLKEKN IMDLYVEKKA101 QKPFLFFHNK EGSTSVFQSV SYPGWFIATS TTSGQPIFLT KERGITNNTN 151 FYLDSVE*

[0026] The discovery of the IL-1 eta polynucleotides of the inventionenables the construction of expression vectors comprising polynucleotidesequences encoding the respective polypeptides and host cellstransfected or transformed with the expression vectors. The inventionalso enables the isolation and purification of biologically active IL-1eta polypeptides and fragments thereof. In yet another embodiment, thepolynucleotides or oligonucleotides thereof can be used as probes toidentify DNA encoding proteins having activities associated with IL-1family members. In addition, the polynucleotides or oligonucleotidesthereof of the present invention may be used to identify humanchromosome 2. Similarly, the polynucleotides or oligonucleotides thereofof the present invention may be used to map genes on human chromosome 2,and to identify genes associated with certain diseases, syndromes orother human conditions associated with human chromosome 2. Among suchdiseases, syndromes or conditions are glaucoma, ectodermal dysplasia,insulin-dependent diabetes mellitus, wrinkly skin syndrome, T-cellleukemia/lymphoma, and tibial muscular dystrophy. Finally,single-stranded sense or antisense oligonucleotides from thesepolynucleotides can be used to inhibit expression of polynucleotidesencoded by the IL-1 eta.

[0027] Further, and in accordance with the present invention, IL-1 etapolypeptides and/or soluble fragments thereof, can be used to activateand/or inhibit the activation of vascular endothelial cells andlymphocytes, induce and/or inhibit the induction of local tissuedestruction and fever (Janeway et al., 1996), inhibit and/or stimulatemacrophages and vascular endothelial cells to produce IL-6, induceand/or inhibit the induction of prostaglandins, nitric oxide synthetase,and metalloproteinases, and upregulate and/or inhibit the upregulationof molecules on the surface of vascular endothelial cells. In additionthese polypeptides and fragmented peptides can also be used to induceand/or inhibit the induction of inflammatory mediators such astranscription factors NF-κB and AP-1, MAP kinases JNK and p38, COX-2,iNOS, and all of the activities stimulated by these molecules. Thepolypeptides of this invention, and fragments thereof, can be used togenerate antibodies, and the invention includes the use of suchantibodies to purify IL-1 eta polypeptides.

[0028] Polynucleotide Molecules

[0029] In one embodiment, the present invention involves certainpolynucleotides that are free from contaminating endogenous material. Apolynucleotide refers to a DNA molecule in the form of a separatefragment or as a component of a larger polynucleotide construct. Thepolynucleotide molecule has been derived from DNA or RNA isolated atleast once in a quantity that allows identification of its componentnucleotide sequence by standard biochemical methods (such as thoseoutlined in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nded., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)).Such sequences are preferably provided and/or constructed in the form ofan open reading frame uninterrupted by internal non-translatedsequences, or introns, that are typically present in eukaryotic genes.Sequences of non-translated DNA can be present 5′ or 3′ from an openreading frame, where the same do not interfere with manipulation orexpression of the coding region. In one embodiment of the instantinvention, the open reading frame runs from nucleotide 112 to the TAAstop codon.

[0030] Polynucleotide molecules of the invention include DNA in bothsingle-stranded and double-stranded form, as well as the RNA complementthereof. DNA includes, for example, cDNA, genomic DNA, chemicallysynthesized DNA, DNA amplified by PCR, and combinations thereof. GenomicDNA may be isolated by conventional techniques, e.g., using the cDNA ofSEQ ID NO:1, or a suitable fragment thereof, as a probe.

[0031] The DNA molecules of the invention include full length genes aswell as polynucleotides and fragments thereof. The full length gene mayinclude the N-terminal signal peptide. Other embodiments include DNAencoding a soluble form, e.g., encoding the extracellular domain of theprotein, either with or without the signal peptide.

[0032] The polynucleotides of the invention are preferentially derivedfrom human sources, but the invention includes those derived fromnon-human species, as well.

[0033] The particularly preferred polynucleotide of the invention hasthe polynucleotide sequence shown in the encoding region of SEQ ID NO:1(beginning at nucleotide 112), for IL-1 eta. cDNA clones having thenucleotide sequence of SEQ ID NO:1 were isolated as described inExample 1. The polypeptide encoded by the IL-1 eta DNA of SEQ ID NO:1 isshown in SEQ ID NO:2.

[0034] The polypeptide of SEQ ID NO:2 shares homology with other IL-1family members.

[0035] Due to the known degeneracy of the genetic code, wherein morethan one codon can encode the same amino acid, a DNA can vary from thatshown in SEQ ID NO:1, and still encode a polypeptide having the aminoacid sequence of SEQ ID NO:2. Such variant DNA sequences can result fromsilent mutations (e.g., occurring during PCR amplification), or can bethe product of deliberate mutagenesis of a native sequence.

[0036] The invention thus provides isolated DNA sequences encodingpolypeptides of the invention, selected from: (a) DNA comprising thenucleotide resides 112-585 of SEQ ID NO:1; (b) DNA encoding thepolypeptides of SEQ ID NO:2; (c) DNA that is the complement of DNA thatis capable of hybridization to a DNA of (a) or (b) under conditions ofmoderate stringency and which encodes polypeptides of the invention; (d)DNA that is the complement of DNA capable of hybridization to a DNA of(a) or (b) under conditions of high stringency and which encodespolypeptides of the invention, and (e) DNA which is degenerate, as aresult of the genetic code, to a DNA defined in (a), (b), (c), or (d)and which encode polypeptides of the invention. Of course, polypeptidesencoded by such DNA sequences are encompassed by the invention.

[0037] As used herein, conditions of moderate stringency can be readilydetermined by those having ordinary skill in the art based on, forexample, the length of the DNA. The basic conditions are set forth bySambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1,pp. 1.101-104, Cold Spring Harbor Laboratory Press, (1989), and includeuse of a prewashing solution for the nitrocellulose filters 5×SSC, 0.5%SDS, 1.0 mM EDTA (pH 8.0), hybridization conditions of about 50%formamide, 6×SSC at about 42° C. (or other similar hybridizationsolution, such as Stark's solution, in about 50% formamide at about 42°C.), and washing conditions of about 60° C., 0.5×SSC, 0.1% SDS.Conditions of high stringency can also be readily determined by theskilled artisan based on, for example, the length of the DNA. Generally,such conditions are defined as hybridization conditions as above, andwith washing at approximately 68° C., 0.2×SSC, 0.1% SDS. The skilledartisan will recognize that the temperature and wash solution saltconcentration can be adjusted as necessary according to factors such asthe length of the probe.

[0038] Also included as an embodiment of the invention is DNA encodingpolypeptide fragments and polypeptides comprising inactivatedN-glycosylation site(s), inactivated protease processing site(s), orconservative amino acid substitution(s), as described below.

[0039] In another embodiment, the polynucleotide molecules of theinvention also comprise nucleotide sequences that are at least 80%identical to a native sequence. Also contemplated are embodiments inwhich a polynucleotide molecule comprises a sequence that is at least90% identical, at least 95% identical, at least 98% identical, at least99% identical, or at least 99.9% identical to a native sequence.

[0040] The percent identity may be determined by visual inspection andmathematical calculation. Alternatively, the percent identity of twopolynucleotide sequences can be determined by comparing sequenceinformation using the GAP computer program, version 6.0 described byDevereux et al. (Nucl. Acids Res. 12:387, 1984) and available from theUniversity of Wisconsin Genetics Computer Group (UWGCG). The preferreddefault parameters for the GAP program include: (1) a unary comparisonmatrix (containing a value of 1 for identities and 0 for non-identities)for nucleotides, and the weighted comparison matrix of Gribskov andBurgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz andDayhoff, eds., Atlas of Protein Sequence and Structure, NationalBiomedical Research Foundation, pp. 353-358, 1979; (2) a penalty of 3.0for each gap and an additional 0.10 penalty for each symbol in each gap;and (3) no penalty for end gaps. Other programs used by one skilled inthe art of sequence comparison may also be used.

[0041] The invention provides isolated polynucleotides useful in theproduction of polypeptides. Such polypeptides may be prepared by any ofa number of conventional techniques. A DNA sequence encoding apolypeptide of the invention, or desired fragment thereof may besubcloned into an expression vector for production of the polypeptide orfragment. The DNA sequence advantageously is fused to a sequenceencoding a suitable leader or signal peptide. Alternatively, the desiredfragment may be chemically synthesized using known techniques. DNAfragments also may be produced by restriction endonuclease digestion ofa full length cloned DNA sequence, and isolated by electrophoresis onagarose gels. If necessary, oligonucleotides that reconstruct the 5′ or3′ terminus to a desired point may be ligated to a DNA fragmentgenerated by restriction enzyme digestion. Such oligonucleotides mayadditionally contain a restriction endonuclease cleavage site upstreamof the desired coding sequence, and position an initiation codon (ATG)at the N-terminus of the coding sequence.

[0042] The well-known polymerase chain reaction (PCR) procedure also maybe employed to isolate and amplify a DNA sequence encoding a desiredprotein fragment. Oligonucleotides that define the desired termini ofthe DNA fragment are employed as 5′ and 3′ primers. The oligonucleotidesmay additionally contain recognition sites for restrictionendonucleases, to facilitate insertion of the amplified DNA fragmentinto an expression vector. PCR techniques are described in Saiki et al.,Science 239:487 (1988); Recombinant DNA Methodology, Wu et al., eds.,Academic Press, Inc., San Diego (1989), pp. 189-196; and PCR Protocols:A Guide to Methods and Applications, Innis et al., eds., Academic Press,Inc. (1990).

[0043] Polypeptides and Fragments Thereof

[0044] The invention encompasses polypeptides and fragments thereof invarious forms, including those that are naturally occurring or producedthrough various techniques such as procedures involving recombinant DNAtechnology. Such forms include, but are not limited to, derivatives,variants, and oligomers, as well as fusion proteins or fragmentsthereof.

[0045] The polypeptides of the invention include full length proteinsencoded by the polynucleotide sequences set forth above. Particularlypreferred polypeptides of IL-1 eta comprise the amino acid sequence ofSEQ ID NO:2.

[0046] The polypeptides of the invention may be secreted and, thus,soluble. Soluble polypeptides are capable of being secreted from thecells in which they are expressed. In general, soluble polypeptides maybe identified (and distinguished from non-soluble membrane-boundcounterparts) by separating intact cells which express the desiredpolypeptide from the culture medium, e.g., by centrifugation, andassaying the medium (supernatant) for the presence of the desiredpolypeptide. The presence of polypeptide in the medium indicates thatthe polypeptide was secreted from the cells and thus is a soluble formof the protein.

[0047] In one embodiment, the soluble polypeptides and fragments thereofcomprise all or part of the extracellular domain, but lack thetransmembrane region that would cause retention of the polypeptide on acell membrane. A soluble polypeptide may include the cytoplasmic domain,or a portion thereof, as long as the polypeptide is secreted from thecell in which it is produced.

[0048] In general, the use of soluble forms is advantageous for certainapplications. Purification of the polypeptides from recombinant hostcells is facilitated, since the soluble polypeptides are secreted fromthe cells. Further, soluble polypeptides are generally more suitable forintravenous administration.

[0049] The invention also provides polypeptides and fragments of theextracellular domain that retain a desired biological activity.Particular embodiments are directed to polypeptide fragments of SEQ IDNO:2 that retain the ability to bind the native cognates, substrates, orcounter-structure (“binding partner”). Such a fragment may be a solublepolypeptide, as described above. In another embodiment, the polypeptidesand fragments advantageously include regions that are conserved in theIL-1 ligand and IL-1 receptor family as described above.

[0050] Also provided herein are polypeptide fragments comprising atleast 20, or at least 30, contiguous amino acids of the sequences of SEQID NO:2. Polypeptide fragments also may be employed as immunogens, ingenerating antibodies.

[0051] Naturally occurring variants as well as derived variants of thepolypeptides and fragments are provided herein. Variants may exhibitamino acid sequences that are at least 80% identical. Also contemplatedare embodiments in which a polypeptide or fragment comprises an aminoacid sequence that is at least 90% identical, at least 95% identical, atleast 98% identical, at least 99% identical, or at least 99.9% identicalto the preferred polypeptide or fragment thereof. Percent identity maybe determined by visual inspection and mathematical calculation.Alternatively, the percent identity of two protein sequences can bedetermined by comparing sequence information using the GAP computerprogram, based on the algorithm of Needleman and Wunsch (J. Mol. Bio.48:443, 1970) and available from the University of Wisconsin GeneticsComputer Group (UWGCG). The preferred default parameters for the GAPprogram include: (1) a scoring matrix, blosum62, as described byHenikoff and Henikoff (Proc. Natl. Acad. Sci. USA 89:10915, 1992); (2) agap weight of 12; (3) a gap length weight of 4; and (4) no penalty forend gaps. Other programs used by one skilled in the art of sequencecomparison may also be used.

[0052] The variants of the invention include, for example, those thatresult from alternate mRNA splicing events or from proteolytic cleavage.Alternate splicing of mRNA may, for example, yield a truncated butbiologically active protein, such as a naturally occurring soluble formof the protein. Variations attributable to proteolysis include, forexample, differences in the N- or C-termini upon expression in differenttypes of host cells, due to proteolytic removal of one or more terminalamino acids from the protein (generally from 1-5 terminal amino acids).Proteins in which differences in amino acid sequence are attributable togenetic polymorphism (allelic variation among individuals producing theprotein) are also contemplated herein.

[0053] Additional variants within the scope of the invention includepolypeptides that may be modified to create derivatives thereof byforming covalent or aggregative conjugates with other chemical moieties,such as glycosyl groups, lipids, phosphate, acetyl groups and the like.Covalent derivatives may be prepared by linking the chemical moieties tofunctional groups on amino acid side chains or at the N-terminus orC-terminus of a polypeptide. Conjugates comprising diagnostic(detectable) or therapeutic agents attached thereto are contemplatedherein, as discussed in more detail below.

[0054] Other derivatives include covalent or aggregative conjugates ofthe polypeptides with other proteins or polypeptides, such as bysynthesis in recombinant culture as N-terminal or C-terminal fusions.Examples of fusion proteins are discussed below in connection witholigomers. Further, fusion proteins can comprise peptides added tofacilitate purification and identification. Such peptides include, forexample, poly-His or the antigenic identification peptides described inU.S. Pat. No. 5,011,912 and in Hopp et al., Bio/Technology 6:1204, 1988.One such peptide is the FLAG® peptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys,which is highly antigenic and provides an epitope reversibly bound by aspecific monoclonal antibody, enabling rapid assay and facilepurification of expressed recombinant protein. A murine hybridomadesignated 4E11 produces a monoclonal antibody that binds the FLAG®peptide in the presence of certain divalent metal cations, as describedin U.S. Pat. No. 5,011,912, hereby incorporated by reference. The 4E11hybridoma cell line has been deposited with the American Type CultureCollection under accession no. HB 9259. Monoclonal antibodies that bindthe FLAG® peptide are available from Eastman Kodak Co., ScientificImaging Systems Division, New Haven, Conn.

[0055] Among the variant polypeptides provided herein are variants ofnative polypeptides that retain the native biological activity or thesubstantial equivalent thereof. One example is a variant that binds withessentially the same binding affinity as does the native form. Bindingaffinity can be measured by conventional procedures, e.g., as describedin U.S. Pat. No. 5,512,457 and as set forth below.

[0056] Variants include polypeptides that are substantially homologousto the native form, but which have an amino acid sequence different fromthat of the native form because of one or more deletions, insertions orsubstitutions. Particular embodiments include, but are not limited to,polypeptides that comprise from one to ten deletions, insertions orsubstitutions of amino acid residues, when compared to a nativesequence.

[0057] A given amino acid may be replaced, for example, by a residuehaving similar physiochemical characteristics. Examples of suchconservative substitutions include substitution of one aliphatic residuefor another, such as Ile, Val, Leu, or Ala for one another;substitutions of one polar residue for another, such as between Lys andArg, Glu and Asp, or Gln and Asn; or substitutions of one aromaticresidue for another, such as Phe, Trp, or Tyr for one another. Otherconservative substitutions, e.g., involving substitutions of entireregions having similar hydrophobicity characteristics, are well known.

[0058] Similarly, the DNAs of the invention include variants that differfrom a native DNA sequence because of one or more deletions, insertionsor substitutions, but that encode a biologically active polypeptide.

[0059] The invention further includes polypeptides of the invention withor without associated native-pattern glycosylation. Polypeptidesexpressed in yeast or mammalian expression systems (e.g., COS-1 or COS-7cells) can be similar to or significantly different from a nativepolypeptide in molecular weight and glycosylation pattern, dependingupon the choice of expression system. Expression of polypeptides of theinvention in bacterial expression systems, such as E. coli, providesnon-glycosylated molecules. Further, a given preparation may includemultiple differentially glycosylated species of the protein. Glycosylgroups can be removed through conventional methods, in particular thoseutilizing glycopeptidase. In general, glycosylated polypeptides of theinvention can be incubated with a molar excess of glycopeptidase(Boehringer Mannheim).

[0060] Correspondingly, similar DNA constructs that encode variousadditions or substitutions of amino acid residues or sequences, ordeletions of terminal or internal residues or sequences are encompassedby the invention. For example, N-glycosylation sites in the polypeptideextracellular domain can be modified to preclude glycosylation, allowingexpression of a reduced carbohydrate analog in mammalian and yeastexpression systems. N-glycosylation sites in eukaryotic polypeptides arecharacterized by an amino acid triplet Asn-X-Y, wherein X is any aminoacid except Pro and Y is Ser or Thr. Appropriate substitutions,additions, or deletions to the nucleotide sequence encoding thesetriplets will result in prevention of attachment of carbohydrateresidues at the Asn side chain. Alteration of a single nucleotide,chosen so that Asn is replaced by a different amino acid, for example,is sufficient to inactivate an N-glycosylation site. Alternatively, theSer or Thr can by replaced with another amino acid, such as Ala. Knownprocedures for inactivating N-glycosylation sites in proteins includethose described in U.S. Pat. No. 5,071,972 and EP 276,846, herebyincorporated by reference.

[0061] In another example of variants, sequences encoding Cys residuesthat are not essential for biological activity can be altered to causethe Cys residues to be deleted or replaced with other amino acids,preventing formation of incorrect intramolecular disulfide bridges uponfolding or renaturation.

[0062] Other variants are prepared by modification of adjacent dibasicamino acid residues, to enhance expression in yeast systems in whichKEX2 protease activity is present. EP 212,914 discloses the use ofsite-specific mutagenesis to inactivate KEX2 protease processing sitesin a protein. KEX2 protease processing sites are inactivated bydeleting, adding or substituting residues to alter Arg-Arg, Arg-Lys, andLys-Arg pairs to eliminate the occurrence of these adjacent basicresidues. Lys-Lys pairings are considerably less susceptible to KEX2cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys represents aconservative and preferred approach to inactivating KEX2 sites.

[0063] Oligomers

[0064] Encompassed by the invention are oligomers or fusion proteinsthat contain IL-1 eta polypeptides. Such oligomers may be in the form ofcovalently linked or non-covalently-linked multimers, including dimers,trimers, or higher oligomers. As noted above, preferred polypeptides aresoluble and thus these oligomers may comprise soluble polypeptides. Inone aspect of the invention, the oligomers maintain the binding abilityof the polypeptide components and provide therefor, bivalent, trivalent,etc., binding sites.

[0065] One embodiment of the invention is directed to oligomerscomprising multiple polypeptides joined via covalent or non-covalentinteractions between peptide moieties fused to the polypeptides. Suchpeptides may be peptide linkers (spacers), or peptides that have theproperty of promoting oligomerization. Leucine zippers and certainpolypeptides derived from antibodies are among the peptides that canpromote oligomerization of the polypeptides attached thereto, asdescribed in more detail below.

[0066] Immunoglobulin-Based Oligomers

[0067] As one alternative, an oligomer is prepared using polypeptidesderived from immunoglobulins. Preparation of fusion proteins comprisingcertain heterologous polypeptides fused to various portions ofantibody-derived polypeptides (including the Fc domain) has beendescribed, e.g., by Ashkenazi et al. (PNAS USA 88:10535, 1991); Byrn etal. (Nature 344:677, 1990); and Hollenbaugh and Aruffo (“Construction ofImmunoglobulin Fusion Proteins”, in Current Protocols in Immunology,Suppl. 4, pages 10.19.1-10.19.11, 1992).

[0068] One embodiment of the present invention is directed to a dimercomprising two fusion proteins created by fusing a polypeptide of theinvention to an Fc polypeptide derived from an antibody. A gene fusionencoding the polypeptide/Fc fusion protein is inserted into anappropriate expression vector. Polypeptide/Fc fusion proteins areexpressed in host cells transformed with the recombinant expressionvector, and allowed to assemble much like antibody molecules, whereuponinterchain disulfide bonds form between the Fc moieties to yielddivalent molecules.

[0069] The term “Fc polypeptide” as used herein includes native andmutein forms of polypeptides made up of the Fc region of an antibodycomprising any or all of the CH domains of the Fc region. Truncatedforms of such polypeptides containing the hinge region that promotesdimerization are also included. Preferred polypeptides comprise an Fcpolypeptide derived from a human IgG1 antibody.

[0070] One suitable Fc polypeptide, described in PCT application WO93/10151, hereby incorporated by reference, is a single chainpolypeptide extending from the N-terminal hinge region to the nativeC-terminus of the Fc region of a human IgG1 antibody. Another useful Fcpolypeptide is the Fc mutein described in U.S. Pat. No. 5,457,035 and inBaum et al., (EMBO J. 13:3992-4001, 1994) incorporated herein byreference. The amino acid sequence of this mutein is identical to thatof the native Fc sequence presented in WO 93/10151, except that aminoacid 19 has been changed from Leu to Ala, amino acid 20 has been changedfrom Leu to Glu, and amino acid 22 has been changed from Gly to Ala. Themutein exhibits reduced affinity for Fc receptors.

[0071] The above-described fusion proteins comprising Fc moieties (andoligomers formed therefrom) offer the advantage of facile purificationby affinity chromatography over Protein A or Protein G columns.

[0072] In other embodiments, the polypeptides of the invention may besubstituted for the variable portion of an antibody heavy or lightchain. If fusion proteins are made with both heavy and light chains ofan antibody, it is possible to form an oligomer with as many as fourpolypeptide extracellular region.

[0073] Alternatively, the oligomer is a fusion protein comprisingmultiple polypeptides, with or without peptide linkers (spacerpeptides). Among the suitable peptide linkers are those described inU.S. Pat. Nos. 4,751,180 and 4,935,233, which are hereby incorporated byreference. A DNA sequence encoding a desired peptide linker may beinserted between, and in the same reading frame as, the DNA sequences ofthe invention, using any suitable conventional technique. For example, achemically synthesized oligonucleotide encoding the linker may beligated between the sequences. In particular embodiments, a fusionprotein comprises from two to four soluble polypeptides of theinvention, separated by peptide linkers.

[0074] Another method for preparing the oligomers of the inventioninvolves use of a leucine zipper. Leucine zipper domains are peptidesthat promote oligomerization of the proteins in which they are found.Leucine zippers were originally identified in several DNA-bindingproteins (Landschulz et al., Science 240:1759, 1988), and have sincebeen found in a variety of different proteins. Among the known leucinezippers are naturally occurring peptides and derivatives thereof thatdimerize or trimerize.

[0075] The zipper domain (also referred to herein as an oligomerizing,or oligomer-forming, domain) comprises a repetitive heptad repeat, oftenwith four or five leucine residues interspersed with other amino acids.Examples of zipper domains are those found in the yeast transcriptionfactor GCN4 and a heat-stable DNA-binding protein found in rat liver(C/EBP; Landschulz et al., Science 243:1681, 1989). Two nucleartransforming proteins, fos and jun, also exhibit zipper domains, as doesthe gene product of the murine proto-oncogene, c-myc (Landschulz et al.,Science 240:1759, 1988). The products of the nuclear oncogenes fos andjun comprise zipper domains that preferentially form heterodimers(O'Shea et al., Science 245:646, 1989, Turner and Tjian, Science243:1689, 1989). The zipper domain is necessary for biological activity(DNA binding) in these proteins.

[0076] The fusogenic proteins of several different viruses, includingparamyxovirus, coronavirus, measles virus and many retroviruses, alsopossess zipper domains (Buckland and Wild, Nature 338:547,1989; Britton,Nature 353:394, 1991; Delwart and Mosialos, AIDS Research and HumanRetroviruses 6:703, 1990). The zipper domains in these fusogenic viralproteins are near the transmembrane region of the proteins; it has beensuggested that the zipper domains could contribute to the oligomericstructure of the fusogenic proteins. Oligomerization of fusogenic viralproteins is involved in fusion pore formation (Spruce et al, Proc. Natl.Acad. Sci. U.S.A. 88:3523, 1991). Zipper domains have also been reportedrecently to play a role in oligomerization of heat-shock transcriptionfactors (Rabindran et al., Science 259:230, 1993).

[0077] Zipper domains fold as short, parallel coiled coils. (O'Shea etal., Science 254:539; 1991) The general architecture of the parallelcoiled coil has been well characterized, with a “knobs-into-holes”packing as proposed by Crick in 1953 (Acta Crystallogr. 6:689). Thedimer formed by a zipper domain is stabilized by the heptad repeat,designated (abcdefg)_(n) according to the notation of McLachlan andStewart (J. Mol. Biol. 98:293; 1975), in which residues a and d aregenerally hydrophobic residues, with d being a leucine, which line up onthe same face of a helix. Oppositely-charged residues commonly occur atpositions g and e. Thus, in a parallel coiled coil formed from twohelical zipper domains, the “knobs” formed by the hydrophobic sidechains of the first helix are packed into the “holes” formed between theside chains of the second helix.

[0078] The residues at position d (often leucine) contribute largehydrophobic stabilization energies, and are important for oligomerformation (Krystek: et al., Int. J. Peptide Res. 38:229, 1991). Lovejoyet al. (Science 259:1288, 1993) recently reported the synthesis of atriple-stranded α-helical bundle in which the helices run up-up-down.Their studies confirmed that hydrophobic stabilization energy providesthe main driving force for the formation of coiled coils from helicalmonomers. These studies also indicate that electrostatic interactionscontribute to the stoichiometry and geometry of coiled coils. Furtherdiscussion of the structure of leucine zippers is found in Harbury etal. (Science 262:1401, 26 November 1993)

[0079] Examples of leucine zipper domains suitable for producing solubleoligomeric proteins are described in PCT application WO 94/10308, andthe leucine zipper derived from lung surfactant protein D (SPD)described in Hoppe et al. (FEBS Letters 344:191, 1994), herebyincorporated by reference. The use of a modified leucine zipper thatallows for stable trimerization of a heterologous protein fused theretois described in Fanslow et al. (Semin. Immunol. 6:267-278, 1994).Recombinant fusion proteins comprising a soluble polypeptide fused to aleucine zipper peptide are expressed in suitable host cells, and thesoluble oligomer that forms is recovered from the culture supernatant.

[0080] Certain leucine zipper moieties preferentially form trimers. Oneexample is a leucine zipper derived from lung surfactant protein D(SPD), as described in Hoppe et al. (FEBS Letters 344:191, 1994) and inU.S. Pat. No. 5,716,805, hereby incorporated by reference in theirentirety. This lung SPD-derived leucine zipper peptide comprises theamino acid sequence Pro Asp Val Ala Ser Leu Arg Gln Gln Val Glu Ala LeuGln Gly Gln Val Gln His Leu Gln Ala Ala Phe Ser Gln Tyr.

[0081] Another example of a leucine zipper that promotes trimerizationis a peptide comprising the amino acid sequence Arg Met Lys Gln Ile GluAsp Lys Ile Glu Glu Ile Leu Ser Lys Ile Tyr His Ile Glu Asn Glu Ile AlaArg Ile Lys Lys Leu Ile Gly Glu Arg, as described in U.S. Pat. No.5,716,805. In one alternative embodiment, an N-terminal Asp residue isadded; in another, the peptide lacks the N-terminal Arg residue.

[0082] Fragments of the foregoing zipper peptides that retain theproperty of promoting oligomerization may be employed as well. Examplesof such fragments include, but are not limited to, peptides lacking oneor two of the N-terminal or C-terminal residues presented in theforegoing amino acid sequences. Leucine zippers may be derived fromnaturally occurring leucine zipper peptides, e.g., via conservativesubstitution(s) in the native amino acid sequence, wherein the peptide'sability to promote oligomerization is retained.

[0083] Other peptides derived from naturally occurring trimeric proteinsmay be employed in preparing trimeric oligomers. Alternatively,synthetic peptides that promote oligomerization may be employed. Inparticular embodiments, leucine residues in a leucine zipper moiety arereplaced by isoleucine residues. Such peptides comprising isoleucine maybe referred to as isoleucine zippers, but are encompassed by the term“leucine zippers” as employed herein.

[0084] Production of Polypeptides and Fragments Thereof

[0085] Expression, isolation and purification of the polypeptides andfragments of the invention may be accomplished by any suitabletechnique, including but not limited to the following:

[0086] Expression Systems

[0087] The present invention also provides recombinant cloning andexpression vectors containing DNA, as well as host cell containing therecombinant vectors. Expression vectors comprising DNA may be used toprepare the polypeptides or fragments of the invention encoded by theDNA. A method for producing polypeptides comprises culturing host cellstransformed with a recombinant expression vector encoding thepolypeptide, under conditions that promote expression of thepolypeptide, then recovering the expressed polypeptides from theculture. The skilled artisan will recognize that the procedure forpurifying the expressed polypeptides will vary according to such factorsas the type of host cells employed, and whether the polypeptide ismembrane-bound or a soluble form that is secreted from the host cell.

[0088] Any suitable expression system may be employed. The vectorsinclude a DNA encoding a polypeptide or fragment of the invention,operably linked to suitable transcriptional or translational regulatorynucleotide sequences, such as those derived from a mammalian, microbial,viral, or insect gene. Examples of regulatory sequences includetranscriptional promoters, operators, or enhancers, an mRNA ribosomalbinding site, and appropriate sequences which control transcription andtranslation initiation and termination. Nucleotide sequences areoperably linked when the regulatory sequence functionally relates to theDNA sequence. Thus, a promoter nucleotide sequence is operably linked toa DNA sequence if the promoter nucleotide sequence controls thetranscription of the DNA sequence. An origin of replication that confersthe ability to replicate in the desired host cells, and a selection geneby which transformants are identified, are generally incorporated intothe expression vector.

[0089] In addition, a sequence encoding an appropriate signal peptide(native or heterologous) can be incorporated into expression vectors. ADNA sequence for a signal peptide (secretory leader) may be fused inframe to the polynucleotide sequence of the invention so that the DNA isinitially transcribed, and the mRNA translated, into a fusion proteincomprising the signal peptide. A signal peptide that is functional inthe intended host cells promotes extracellular secretion of thepolypeptide. The signal peptide is cleaved from the polypeptide uponsecretion of polypeptide from the cell.

[0090] The skilled artisan will also recognize that the position(s) atwhich the signal peptide is cleaved may differ from that predicted bycomputer program, and may vary according to such factors as the type ofhost cells employed in expressing a recombinant polypeptide. A proteinpreparation may include a mixture of protein molecules having differentN-terminal amino acids, resulting from cleavage of the signal peptide atmore than one site.

[0091] Suitable host cells for expression of polypeptides includeprokaryotes, yeast or higher eukaryotic cells. Mammalian or insect cellsare generally preferred for use as host cells. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are described, for example, in Pouwels et al. CloningVectors: A Laboratory Manual, Elsevier, New York, (1985). Cell-freetranslation systems could also be employed to produce polypeptides usingRNAs derived from DNA constructs disclosed herein.

[0092] Prokaryotic Systems

[0093] Prokaryotes include gram-negative or gram-positive organisms.Suitable prokaryotic host cells for transformation include, for example,E. coli, Bacillus subtilis, Salmonella typhimurium, and various otherspecies within the genera Pseudomonas, Streptomyces, and Staphylococcus.In a prokaryotic host cell, such as E. coli, a polypeptide may includean N-terminal methionine residue to facilitate expression of therecombinant polypeptide in the prokaryotic host cell. The N-terminal Metmay be cleaved from the expressed recombinant polypeptide.

[0094] Expression vectors for use in prokaryotic host cells generallycomprise one or more phenotypic selectable marker genes. A phenotypicselectable marker gene is, for example, a gene encoding a protein thatconfers antibiotic resistance or that supplies an autotrophicrequirement. Examples of useful expression vectors for prokaryotic hostcells include those derived from commercially available plasmids such asthe cloning vector pBR322 (ATCC 37017). pBR322 contains genes forampicillin and tetracycline resistance and thus provides simple meansfor identifying transformed cells. An appropriate promoter and a DNAsequence are inserted into the pBR322 vector. Other commerciallyavailable vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wis.,USA).

[0095] Promoter sequences commonly used for recombinant prokaryotic hostcell expression vectors include β-lactamase (penicillinase), lactosepromoter system (Chang et al., Nature 275:615, 1978; and Goeddel et al.,Nature 281:544, 1979), tryptophan (trp) promoter system (Goeddel et al.,Nucl. Acids Res. 8:4057, 1980; and EP-A-36776) and tac promoter(Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, p. 412, 1982). A particularly useful prokaryotic host cellexpression system employs a phage λP_(L) promoter and a cI857tsthermolabile repressor sequence. Plasmid vectors available from theAmerican Type Culture Collection which incorporate derivatives of theλP_(L) promoter include plasmid pHUB2 (resident in E. coli strain JMB9,ATCC 37092) and pPLc28 (resident in E. coli RR1, ATCC 53082).

[0096] Yeast Systems

[0097] Alternatively, the polypeptides may be expressed in yeast hostcells, preferably from the Saccharomyces genus (e.g., S. cerevisiae).Other genera of yeast, such as Pichia or Kluyveromyces, may also beemployed. Yeast vectors will often contain an origin of replicationsequence from a 2μ yeast plasmid, an autonomously replicating sequence(ARS), a promoter region, sequences for polyadenylation, sequences fortranscription termination, and a selectable marker gene. Suitablepromoter sequences for yeast vectors include, among others, promotersfor metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J.Biol. Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J.Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900,1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phospho-glucose isomerase, andglucokinase. Other suitable vectors and promoters for use in yeastexpression are further described in Hitzeman, EPA-73,657. Anotheralternative is the glucose-repressible ADH2 promoter described byRussell et al. (J. Biol. Chem. 258:2674, 1982) and Beier et al. (Nature300:724, 1982). Shuttle vectors replicable in both yeast and E. coli maybe constructed by inserting DNA sequences from pBR322 for selection andreplication in E. coli (Amp^(r) gene and origin of replication) into theabove-described yeast vectors.

[0098] The yeast α-factor leader sequence may be employed to directsecretion of the polypeptide. The α-factor leader sequence is ofteninserted between the promoter sequence and the structural gene sequence.See, e.g., Kurjan et al., Cell 30:933, 1982 and Bitter et al., Proc.Natl. Acad. Sci. USA 81:5330, 1984. Other leader sequences suitable forfacilitating secretion of recombinant polypeptides from yeast hosts areknown to those of skill in the art. A leader sequence may be modifiednear its 3′ end to contain one or more restriction sites. This willfacilitate fusion of the leader sequence to the structural gene.

[0099] Yeast transformation protocols are known to those of skill in theart. One such protocol is described by Hinnen et al., Proc. Natl. Acad.Sci. USA 75:1929, 1978. The Hinnen et al. protocol selects for Trp⁺transformants in a selective medium, wherein the selective mediumconsists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose,10 mg/ml adenine and 20 mg/ml uracil.

[0100] Yeast host cells transformed by vectors containing an ADH2promoter sequence may be grown for inducing expression in a “rich”medium. An example of a rich medium is one consisting of 1% yeastextract, 2% peptone, and 1% glucose supplemented with 80 mg/ml adenineand 80 mg/ml uracil. Derepression of the ADH2 promoter occurs whenglucose is exhausted from the medium.

[0101] Mammalian or Insect Systems

[0102] Mammalian or insect host cell culture systems also may beemployed to express recombinant polypeptides. Bacculovirus systems forproduction of heterologous proteins in insect cells are reviewed byLuckow and Summers, Bio/Technology 6:47 (1988). Established cell linesof mammalian origin also may be employed. Examples of suitable mammalianhost cell lines include the COS-7 line of monkey kidney cells (ATCC CRL1651) (Gluzman et al., Cell 23:175, 1981), L cells, C127 cells, 3T3cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, andBHK (ATCC CRL 10) cell lines, and the CV1/EBNA cell line derived fromthe African green monkey kidney cell line CV1 (ATCC CCL 70) as describedby McMahan et al. (EMBO J. 10: 2821, 1991).

[0103] Established methods for introducing DNA into mammalian cells havebeen described (Kaufman, R. J., Large Scale Mammalian Cell Culture,1990, pp. 15-69). Additional protocols using commercially availablereagents, such as Lipofectamine lipid reagent (Gibco/BRL) orLipofectamine-Plus lipid reagent, can be used to transfect cells(Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417, 1987). Inaddition, electroporation can be used to transfect mammalian cells usingconventional procedures, such as those in Sambrook et al. (MolecularCloning: A Laboratory Manual, 2 ed. Vol. 1-3, Cold Spring HarborLaboratory Press, 1989). Selection of stable transformants can beperformed using methods known in the art, such as, for example,resistance to cytotoxic drugs. Kaufman et al., Meth. in Enzymology185:487-511, 1990, describes several selection schemes, such asdihydrofolate reductase (DHFR) resistance. A suitable host strain forDHFR selection can be CHO strain DX-B11, which is deficient in DHFR(Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:42164220, 1980). Aplasmid expressing the DHFR cDNA can be introduced into strain DX-B11,and only cells that contain the plasmid can grow in the appropriateselective media. Other examples of selectable markers that can beincorporated into an expression vector include cDNAs conferringresistance to antibiotics, such as G418 and hygromycin B. Cellsharboring the vector can be selected on the basis of resistance to thesecompounds.

[0104] Transcriptional and translational control sequences for mammalianhost cell expression vectors can be excised from viral genomes. Commonlyused promoter sequences and enhancer sequences are derived from polyomavirus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus.DNA sequences derived from the SV40 viral genome, for example, SV40origin, early and late promoter, enhancer, splice, and polyadenylationsites can be used to provide other genetic elements for expression of astructural gene sequence in a mammalian host cell. Viral early and latepromoters are particularly useful because both are easily obtained froma viral genome as a fragment, which can also contain a viral origin ofreplication (Fiers et al., Nature 273:113, 1978; Kaufman, Meth. inEnzymology, 1990). Smaller or larger SV40 fragments can also be used,provided the approximately 250 bp sequence extending from the Hind IIIsite toward the Bgl I site located in the SV40 viral origin ofreplication site is included.

[0105] Additional control sequences shown to improve expression ofheterologous genes from mammalian expression vectors include suchelements as the expression augmenting sequence element (EASE) derivedfrom CHO cells (Morris et al., Animal Cell Technology, 1997, pp. 529-534and PCT Application WO 97/25420) and the tripartite leader (TPL) and VAgene RNAs from Adenovirus 2 (Gingeras et al., J. Biol. Chem.257:13475-13491, 1982). The internal ribosome entry site (IRES)sequences of viral origin allows dicistronic mRNAs to be translatedefficiently (Oh and Sarnow, Current Opinion in Genetics and Development3:295-300, 1993; Ramesh et al., Polynucleotides Research 24:2697-2700,1996). Expression of a heterologous cDNA as part of a dicistronic mRNAfollowed by the gene for a selectable marker (e.g. DHFR) has been shownto improve transfectability of the host and expression of theheterologous cDNA (Kaufman, Meth. in Enzymology, 1990). Exemplaryexpression vectors that employ dicistronic mRNAs are pTR-DC/GFPdescribed by Mosser et al., Biotechniques 22:150-161, 1997, and p2A5Idescribed by Morris et al., Animal Cell Technology, 1997, pp. 529-534.

[0106] A useful high expression vector, pCAVNOT, has been described byMosley et al., Cell 59:335-348, 1989. Other expression vectors for usein mammalian host cells can be constructed as disclosed by Okayama andBerg (Mol. Cell. Biol. 3:280, 1983). A useful system for stable highlevel expression of mammalian cDNAs in C127 murine mammary epithelialcells can be constructed substantially as described by Cosman et al.(Mol. Immunol. 23:935, 1986). A useful high expression vector, PMLSVN1/N4, described by Cosman et al., Nature 312:768, 1984, has beendeposited as ATCC 39890. Additional useful mammalian expression vectorsare described in EP-A-0367566, and in WO 91/18982, incorporated byreference herein. In yet another alternative, the vectors can be derivedfrom retroviruses.

[0107] Another useful expression vector, pFLAG®, can be used. FLAG®technology is centered on the fusion of a low molecular weight (1 kD),hydrophilic, FLAG® marker peptide to the N-terminus of a recombinantprotein expressed by pFLAG® expression vectors.

[0108] Regarding signal peptides that may be employed, the native signalpeptide may be replaced by a heterologous signal peptide or leadersequence, if desired. The choice of signal peptide or leader may dependon factors such as the type of host cells in which the recombinantpolypeptide is to be produced. To illustrate, examples of heterologoussignal peptides that are functional in mammalian host cells include thesignal sequence for interleukin-7 (IL-7) described in U.S. Pat. No.4,965,195; the signal sequence for interleukin-2 receptor described inCosman et al., Nature 312:768 (1984); the interleukin-4 receptor signalpeptide described in EP 367,566; the type I interleukin-1 receptorsignal peptide described in U.S. Pat. No. 4,968,607; and the type IIinterleukin-1 receptor signal peptide described in EP 460,846.

[0109] Isolation and Purification

[0110] The “isolated” polypeptides or fragments thereof encompassed bythis invention are polypeptides or fragments that are not in anenvironment identical to an environment in which it or they can be foundin nature. The “purified” polypeptides or fragments thereof encompassedby this invention are essentially free of association with otherproteins or polypeptides, for example, as a purification product ofrecombinant expression systems such as those described above or as apurified product from a non-recombinant source such as naturallyoccurring cells and/or tissues.

[0111] In one preferred embodiment, the purification of recombinantpolypeptides or fragments can be accomplished using fusions ofpolypeptides or fragments of the invention to another polypeptide to aidin the purification of polypeptides or fragments of the invention. Suchfusion partners can include the poly-His or other antigenicidentification peptides described above as well as the Fc moietiesdescribed previously.

[0112] With respect to any type of host cell, as is known to the skilledartisan, procedures for purifying a recombinant polypeptide or fragmentwill vary according to such factors as the type of host cells employedand whether or not the recombinant polypeptide or fragment is secretedinto the culture medium.

[0113] In general, the recombinant polypeptide or fragment can beisolated from the host cells if not secreted, or from the medium orsupernatant if soluble and secreted, followed by one or moreconcentration, salting-out, ion exchange, hydrophobic interaction,affinity purification or size exclusion chromatography steps. As tospecific ways to accomplish these steps, the culture medium first can beconcentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. Following the concentration step, the concentrate can be appliedto a purification matrix such as a gel filtration medium. Alternatively,an anion exchange resin can be employed, for example, a matrix orsubstrate having pendant diethylaminoethyl (DEAE) groups. The matricescan be acrylamide, agarose, dextran, cellulose or other types commonlyemployed in protein purification. Alternatively, a cation exchange stepcan be employed. Suitable cation exchangers include various insolublematrices comprising sulfopropyl or carboxymethyl groups. In addition, achromatofocusing step can be employed. Alternatively, a hydrophobicinteraction chromatography step can be employed. Suitable matrices canbe phenyl or octyl moieties bound to resins. In addition, affinitychromatography with a matrix which selectively binds the recombinantprotein can be employed. Examples of such resins employed are lectincolumns, dye columns, and metal-chelating columns. Finally, one or morereverse-phase high performance liquid chromatography (RP-HPLC) stepsemploying hydrophobic RP-HPLC media, (e.g., silica gel or polymer resinhaving pendant methyl, octyl, octyldecyl or other aliphatic groups) canbe employed to further purify the polypeptides. Some or all of theforegoing purification steps, in various combinations, are well knownand can be employed to provide an isolated and purified recombinantprotein.

[0114] It is also possible to utilize an affinity column comprising apolypeptide-binding protein of the invention, such as a monoclonalantibody generated against polypeptides of the invention, toaffinity-purify expressed polypeptides. These polypeptides can beremoved from an affinity column using conventional techniques, e.g., ina high salt elution buffer and then dialyzed into a lower salt bufferfor use or by changing pH or other components depending on the affinitymatrix utilized, or be competitively removed using the naturallyoccurring substrate of the affinity moiety, such as a polypeptidederived from the invention.

[0115] In this aspect of the invention, polypeptide-binding proteins,such as the anti-polypeptide antibodies of the invention or otherproteins that may interact with the polypeptide of the invention, can bebound to a solid phase support such as a column chromatography matrix ora similar substrate suitable for identifying, separating, or purifyingcells that express polypeptides of the invention on their surface.Adherence of polypeptide-binding proteins of the invention to a solidphase contacting surface can be accomplished by any means. For example,magnetic microspheres can be coated with these polypeptide-bindingproteins and held in the incubation vessel through a magnetic field.Suspensions of cell mixtures are contacted with the solid phase that hassuch polypeptide-binding proteins thereon. Cells having polypeptides ofthe invention on their surface bind to the fixed polypeptide-bindingprotein and unbound cells then are washed away. This affinity-bindingmethod is useful for purifying, screening, or separating suchpolypeptide-expressing cells from solution. Methods of releasingpositively selected cells from the solid phase are known in the art andencompass, for example, the use of enzymes. Such enzymes are preferablynon-toxic and non-injurious to the cells and are preferably directed tocleaving the cell-surface binding partner.

[0116] Alternatively, mixtures of cells suspected of containingpolypeptide-expressing cells of the invention first are incubated with abiotinylated polypeptide-binding protein of the invention. Incubationperiods are typically at least one hour in duration to ensure sufficientbinding to polypeptides of the invention. The resulting mixture then ispassed through a column packed with avidin-coated beads, whereby thehigh affinity of biotin for avidin provides the binding of thepolypeptide-binding cells to the beads. Use of avidin-coated beads isknown in the art. See Berenson, et al. J. Cell. Biochem., 10D:239(1986). Wash of unbound material and the release of the bound cells isperformed using conventional methods.

[0117] The desired degree of purity depends on the intended use of theprotein. A relatively high degree of purity is desired when thepolypeptide is to be administered in vivo, for example. In such a case,the polypeptides are purified such that no protein bands correspondingto other proteins are detectable upon analysis by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE). It will be recognized by one skilled in thepertinent field that multiple bands corresponding to the polypeptide maybe visualized by SDS-PAGE, due to differential glycosylation,differential post-translational processing, and the like. Mostpreferably, the polypeptide of the invention is purified to substantialhomogeneity, as indicated by a single protein band upon analysis bySDS-PAGE. The protein band may be visualized by silver staining,Coomassie blue staining, or (if the protein is radiolabeled) byautoradiography.

[0118] Screening Assays

[0119] The purified polypeptides of the invention (including proteins,polypeptides, fragments, variants, oligomers, and other forms) may betested for the ability to bind the binding partner in any suitableassay, such as a conventional binding assay. The polypeptide may belabeled with a detectable reagent (e.g., a radionuclide, chromophore,enzyme that catalyzes a colorimetric or fluorometric reaction, and thelike). The labeled polypeptide is contacted with cells expressing thebinding partner. The cells then are washed to remove unbound labeledpolypeptide, and the presence of cell-bound label is determined by asuitable technique, chosen according to the nature of the label.

[0120] Another type of suitable binding assay is a competitive bindingassay. Competitive binding assays can be performed by conventionalmethodology. Reagents that may be employed in competitive binding assaysinclude radiolabeled polypeptides of the invention and intact cellsexpressing the binding partner (endogenous or recombinant). For example,a radiolabeled soluble IL-1 eta fragment can be used to compete with asoluble IL-1 eta variant for binding to cell surface IL-1 eta receptors.Instead of intact cells, one could substitute a soluble bindingpartner/Fc fusion protein bound to a solid phase through the interactionof Protein A or Protein G (on the solid phase) with the Fc moiety.Chromatography columns that contain Protein A and Protein G includethose available from Pharmacia Biotech, Inc., Piscataway, N.J.

[0121] Another type of competitive binding assay utilizes radiolabeledsoluble binding partner, such as a soluble IL-1 eta receptor/Fc fusionprotein, and intact cells expressing the binding partner. Qualitativeresults can be obtained by competitive autoradiographic plate bindingassays, while Scatchard plots (Scatchard, Ann. N.Y. Acad. Sci. 51:660,1949) may be utilized to generate quantitative results. Such bindingassays may be useful in evaluating the biological activity of a variantpolypeptide by assaying for the variant's ability to compete with thenative protein for binding to the binding partner.

[0122] The IL-1 eta polypeptide of the present invention may also beused in a screening assay for compounds and small molecules whichinhibit activation by (antagonize) the IL-1 eta polypeptide of theinstant invention. Thus, polypeptides of the invention may be used toidentify antagonists from, for example, cells, cell-free preparations,chemical libraries, and natural product mixtures. The antagonists may benatural or modified substrates, ligands, enzymes, receptors, etc. of theIL-1 eta polypeptide, or may be structural or functional mimetics of theIL-1 eta polypeptide. The antagonists may further be small molecules,peptides, antibodies and antisense oligonucleotides.

[0123] One embodiment of a method for identifying compounds whichantagonize the IL-1 eta polypeptide is contacting a candidate compoundwith cells which respond to IL-1 eta polypeptide and observe the bindingof IL-1 eta to the cells, or stimulation or inhibition of a functionalresponse. The activity of the cells which were contacted with thecandidate compound could then be compared with the identical cells whichwere not contacted for IL-1 eta polypeptide activity and IL-1 etapolypeptide agonists and antagonists could be identified. A stillfurther embodiment of the instant invention provides a method ofidentifying compounds that inhibit the synthesis or secretion of IL-1eta by contacting the candidate compound with cells which express IL-1eta polypeptide and measuring the IL-1 eta production. The measurementof IL-1 eta production could be performed by a number of well-knownmethods such as measuring the amount of protein present (e.g. an ELISA)or of the protein's activity.

[0124] Drug Discovery

[0125] The purified polypeptides according to the invention willfacilitate the discovery of inhibitors (or antagonists) and/or agonistsof such polypeptides. The use of a purified polypeptide of the inventionin the screening of potential inhibitors and/or agonists thereof isimportant and can eliminate or reduce the possibility of interferingreactions with contaminants.

[0126] In addition, polypeptides of the invention can be used forstructure-based design of polypeptide-inhibitors and/or agonists. Suchstructure-based design is also known as “rational drug design.” Thepolypeptides can be three-dimensionally analyzed by, for example, X-raycrystallography, nuclear magnetic resonance or homology modeling, all ofwhich are well-known methods. The use of the polypeptide structuralinformation in molecular modeling software systems to assist ininhibitor design and inhibitor-polypeptide interaction is alsoencompassed by the invention. Such computer-assisted modeling and drugdesign can utilize information such as chemical conformational analysis,electrostatic potential of the molecules, protein folding, etc. Forexample, most of the design of class-specific inhibitors ofmetalloproteases has focused on attempts to chelate or bind thecatalytic zinc atom. Synthetic inhibitors are usually designed tocontain a negatively-charged moiety to which is attached a series ofother groups designed to fit the specificity pockets of the particularprotease. A particular method of the invention comprises analyzing thethree dimensional structure of polypeptides of the invention for likelybinding sites of substrates, synthesizing a new molecule thatincorporates a predictive reactive site, and assaying the new moleculeas described above.

[0127] Specific screening methods are known in the art and along withintegrated robotic systems and collections of chemical compounds/naturalproducts are extensively incorporated in high throughput screening sothat large numbers of test compounds can be tested for antagonist oragonist activity within a short amount of time. These methods includehomogeneous assay formats such as fluorescence resonance energytransfer, fluorescence polarization, time-resolved fluorescenceresonance energy transfer, scintillation proximity assays, reporter geneassays, fluorescence quenched enzyme substrate, chromogenic enzymesubstrate and electrochemiluminescence, as well as more traditionalheterogeneous assay formats such as enzyme-linked immunosorbant assays(ELISA) or radioimmunoassays. Homogeneous assays are preferred. Alsocomprehended herein are cell-based assays, for example those utilizingreporter genes, as well as functional assays that analyze the effect ofan antagonist or agonist on biological function(s) or activity(ies) ofIL-1 eta (for example, stimulation of the secretion of cytokines orinhibition thereof, as disclosed herein). Moreover, animal models ofinflammatory conditions are useful assays of biological activity.

[0128] Accordingly, in one aspect of the invention, there is provided amethod for screening a test compound to determine whether the testcompound affects (or modulates) a biological activity of an IL-1 etapolypeptide, the method comprising contacting the test compound and theIL-1 eta polypeptide with cells capable of exhibiting the biologicalactivity when contacted with IL-1 eta, and analyzing the cells for theoccurrence of the biological activity, wherein if the biologicalactivity observed in the presence of the test compound differs from thebiological activity that is observed when the test compound is absent,the test compound affects the biological activity of the IL-1 eta. Thecells may be contacted in vitro or in vivo.

[0129] As used herein, the IL-1 eta polypeptide comprises a polypeptideselected from the group consisting of the polypeptides of SEQ ID NO:2,and polypeptides encoded by DNAs that hybridize under moderatelystringent conditions to the DNA of SEQ ID NO:1. Such polypeptidesinclude polypeptides comprising variant amino acid sequences that are atleast 80% identical to the polypeptides of SEQ ID NO:2 (preferably, thevariant amino acid sequences that are at least 90% identical, morepreferably at least 95% identical, most preferably at least 97%identical, to the polypeptides of SEQ ID NO:2). Additional examples ofuseful IL-1 eta polypeptides include polypeptides comprising the aminoacid sequences of SEQ ID NO:2 wherein the polypeptides comprisealterations to the amino acid sequences selected from the groupconsisting of inactivated N-glycosylation site(s), inactivated proteaseprocessing site(s), conservative amino acid substitution(s), andcombinations thereof. Moreover, fragments of the aforesaid polypeptidesthat have at least one activity of IL-1 eta as described below are alsocomprehended herein.

[0130] IL-1 eta biological activity includes, but is not limited to,modulation of cytokine expression, modulation of the expression ofmolecules indicative of activation of an immune or inflammatory response(for example, COX2, iNOS), modulation of cell-surface moleculeexpression, modulation of activation of one or more signaling cascades,modulation of induction of mRNAs for the aforementioned proteins,modulation of induction of cell proliferation and/or cell death,induction of morphological and/or functional changes in cells, andcombinations thereof. The inventive methods comprise methods of assayingfor any of these biological activities. Those of skill in the art willrecognize that modulation of cytokines means that the levels ofexpression of certain cytokines increase while the levels of othercytokines decreases, and that such combinations are comprehended in theterm modulation; the same is true for other activities of IL-1 eta.

[0131] When the methods of the present invention include assaying forIL-1 eta modulation of cytokine expression, cytokines that may beassayed include (but are not limited to) IL-1 alpha, IL-1 beta,TNF-alpha, IL-10, IFN-gamma, IL-12 (in particular, the p40 subunit),IL-6, IL-1ra, IL-4, IL-13, GM-CSF, IL-18, IL-1 homologs such as IL-1epsilon, IL-1 eta, IL-1 theta, IL-1 zeta, and IL-1 H1, and combinationsthereof. Similarly, when the screening methods of the present inventioninclude assaying for IL-1 eta modulation of cell surface moleculeexpression, the cell surface molecules that may be assayed includeICAM-1, TLR4, TLR5, TLR9, DC-B7, MHC class I and II antigens, VCAM,ELAM, B7-1, B7-2, CD40L, and combinations thereof.

[0132] IL-1 eta mediated modulation of signaling pathways often involvesa cascade of molecular changes, for example as discussed previouslywherein a receptor propagates a ligand-receptor mediated signal byspecifically activating intracellular kinases which phosphorylate targetsubstrates (which can themselves be kinases that become activatedfollowing phosphorylation, or adaptor molecules that facilitatedown-stream signaling through protein-protein interaction followingphosphorylation), resulting in the activation of other factors (forexample, NFkappaB). When the screening methods of the present inventioninclude assaying for IL-1 eta induced modulation of signaling pathways,the signaling pathways that may be assayed include those involvingactivation of NFkappaB. Assaying for activation signaling cascadesfurther includes detecting phosphorylation of molecules that occursduring the signaling cascade, as in the phosphorylation of IkappaB(including IkappaB degradation assays, and assays for free IkappaB), p38MAP kinase, and Stress-Activated Protein Kinase (SAPK/JNK).

[0133] Moreover, those of skill in the art understand that biologicalactivity(ies) is/are most often induced by the binding of a ligand(i.e., IL-1 eta) to a receptor (counterstructure or binding moiety)present on a cell; accordingly, as previously described, IL-1 etapolypeptides (including IL-1 eta polypeptide fragments) can be used inbinding studies to identify receptor-expressing cells. Such bindingstudies also provide assays useful in the inventive methods. IL-1 etapolypeptides may also be used to clone receptors (or other moleculesthat bind IL-1 eta) and to screen for molecules that blockreceptor/ligand interactions. Those of ordinary skill in the art furtherunderstand that biological activities include cell proliferation, celldeath, and changes in cell morphology and/or function (for example,activation, maturation); assays that evaluate such effects of IL-1 etaare known in the art, and will also be useful in the inventive methods.Moreover, animal models of syndromes and/or conditions, such as thosedisclosed herein, are useful for screening compounds for biologicalactivity, including screening for antagonism (or agonism) of IL-1 eta.

[0134] The inventive methods further encompass performing more than oneassay to discover and/or analyze agonists or antagonists of IL-1 etaactivity (i.e., combination methods). Generally, such methods compriseselecting test compounds that affect a property of IL-1 eta (i.e., anability of IL-1 eta to bind an IL-1 eta counter structure), then testingthe selected compounds for an effect on another property of IL-1 eta(i.e., contacting the selected test compounds and an IL-1 etapolypeptide with cells capable of exhibiting a biological activity whencontacted with IL-1 eta, and determining whether the compounds affectthe biological activity). For example, the inventive methods maycomprise a first assay to determine whether a candidate moleculeinteracts with (binds to) IL-1 eta. In one embodiment, the first assayis in a high throughput format, numerous forms of which are known in theart and disclosed herein. Such an assay will generally comprise thesteps of: contacting test compounds and an IL-1 eta polypeptide with anIL-1 eta counterstructure; determining whether the test compounds affectthe ability of IL-1 eta to bind the counterstructure; and selecting oneor more test compounds that affect the ability of IL-1 eta to bind thecounterstructure. The inventive combination methods further compriseevaluating selected compounds in a second assay, for agonistic orantagonistic effect on biological activity using one or more of theaforementioned assays.

[0135] Alternatively, the inventive combination methods may comprise afirst assay to determine whether a candidate molecule modulates abiological activity of IL-1 eta, as described herein using an in vitroassay or an in vivo assay (for example, an animal model). According tosuch combination methods, molecules that modulate an IL-1 eta biologicalactivity in this manner are selected using one or more of theaforementioned assays for biological activity, and assayed to determinewhether the candidate molecule(s) bind IL-1 eta. The selected moleculesmay be tested to further define the exact region or regions of IL-1 etato which the test molecule binds (for example, epitope mapping forantibodies).

[0136] As disclosed previously, the types of assays for biologicalactivities of IL-1 eta that can be used in the inventive combinationmethods include assays for the expression of cytokines, assays for theexpression of cell-surface molecules, assays to detect activation ofsignaling molecules, assays to detect induction of mRNAs, and assaysthat evaluate cell proliferation or cell death (and combinationsthereof), as described herein. Molecules that bind and that have anagonistic or antagonistic effect on biologic activity will be useful intreating or preventing diseases or conditions with which thepolypeptide(s) are implicated.

[0137] Those of ordinary skill in the art understand that when thebiological activity observed in the presence of the test compound isgreater than that observed when the test compound is absent, the testcompound is an agonist of IL-1 eta, whereas when the biological activityobserved in the presence of the test compound is less than that observedwhen the test compound is absent, the test compound is an antagonist (orinhibitor) of IL-1 eta. Generally, an antagonist will decrease orinhibit, an activity by at least 30%; more preferably, antagonists willinhibit activity by at least 50%, most preferably by at least 90%.Similarly, an agonist will increase, or enhance, an activity by at least20%; more preferably, agonists will enhance activity by at least 30%,most preferably by at least 50%. Those of skill in the art will alsorecognize that agonists and/or antagonists with different levels ofagonism or antagonism respectively may be useful for differentapplications (i.e., for treatment of different disease states).

[0138] Homogeneous assays are mix-and-read style assays that are veryamenable to robotic application, whereas heterogeneous assays requireseparation of free from bound analyte by more complex unit operationssuch as filtration, centrifugation or washing. These assays are utilizedto detect a wide variety of specific biomolecular interactions(including protein-protein, receptor-ligand, enzyme-substrate, and soon), and the inhibition thereof by small organic molecules. These assaymethods and techniques are well known in the art (see, e.g., HighThroughput Screening: The Discovery of Bioactive Substances, John P.Devlin (ed.), Marcel Dekker, New York, 1997 ISBN: 0-8247-0067-8). Thescreening assays of the present invention are amenable to highthroughput screening of chemical libraries and are suitable for theidentification of small molecule drug candidates, antibodies, peptides,and other antagonists and/or agonists, natural or synthetic. Severaluseful assays are disclosed in U.S. Ser. No. 09/851,673, filed May 8,2001 (the relevant disclosure of which is hereby incorporated byreference).

[0139] Candidate Molecules to be Tested:

[0140] The methods of the invention may be used to identify antagonists(also referred to as inhibitors) and agonists of IL-1 eta activity fromcells, cell-free preparations, chemical libraries, cDNA libraries,recombinant antibody libraries (or libraries comprising subunits ofantibodies) and natural product mixtures. The antagonists and agonistsmay be natural or modified substrates, ligands, enzymes, receptors, etc.of the polypeptides of the instant invention, or may be structural orfunctional mimetics of IL-1 eta or its binding partner/counterstructure.Potential antagonists of the instant invention include small molecules,peptides and antibodies that bind to and occupy a binding site of theinventive polypeptides or a binding partner thereof, causing them to beunavailable to bind to their natural binding partners and thereforepreventing normal biological activity. Antagonists also includechemicals (including small molecules and peptides) that interfere withthe signaling pathways used by IL-1 eta (for example, by inhibiting theinteraction of receptor subunits, or inhibiting the interaction ofintracellular components of the signaling cascade). Potential agonistsinclude small molecules, peptides and antibodies which bind to theinstant polypeptides or binding partners thereof, and elicit the same orenhanced biologic effects as those caused by the binding of thepolypeptides of the instant invention. Moreover, substances thatactivate (or enhance) the signaling pathways used by IL-1 eta are alsoincluded within the scope of agonists of IL-1 eta.

[0141] Small molecule agonists and antagonists are usually less than 10Kmolecular weight and may possess a number of physicochemical andpharmacological properties which enhance cell penetration, resistdegradation and prolong their physiological half-lives (Gibbs, J.,Pharmaceutical Research in Molecular Oncology, Cell, Vol. 79 (1994)).Antibodies, which include intact molecules as well as fragments such asFab and F(ab′)2 fragments, as well as recombinant molecules derivedtherefrom (including antibodies expressed on phage, intrabodies, singlechain antibodies such as scFv and other molecules derived fromimmunoglobulins that are known in the art), may be used to bind to andinhibit the polypeptides of the instant invention by blocking thepropagation of a signaling cascade. It is preferable that the antibodiesare humanized, and more preferable that the antibodies are human. Theantibodies of the present invention may be prepared by any of a varietyof well-known methods, as disclosed herein.

[0142] Additional examples of candidate molecules, also referred toherein as “test molecules” or “test compounds,” to be tested for theability to modulate IL-1 eta activity include, but are not limited to,carbohydrates, small molecules (usually organic molecules or peptides),proteins, and nucleic acid molecules (including oligonucleotidefragments typically consisting of from 8 to 30 nucleic acid residues).Peptides to be tested typically consist of from 5 to 25 amino acidresidues. Also, candidate nucleic acid molecules can be antisensenucleic acid sequences, and/or can possess ribozyme activity.

[0143] Small molecules to be screened using the hereindescribedscreening assays can typically be administered orally or by injection toa patient in need thereof. Small molecules that can be administeredorally are especially preferred. The small molecules of the inventionpreferably will not be toxic (or only minimally toxic) at the dosesrequired for them to be effective as pharmaceutical agents, and they arepreferably not subject to rapid loss of activity in the body, such asthe loss of activity that might result from rapid enzymatic or chemicaldegradation. In addition, pharmaceutically useful small molecules arepreferably not immunogenic.

[0144] The methods of the invention can be used to screen for antisensemolecules that inhibit the functional expression of one or more mRNAmolecules that encode one or more proteins that mediate an IL-1eta-dependent cellular response. An anti-sense nucleic acid molecule isa DNA sequence that is capable of can hybridizing to the target mRNAmolecule through Watson-Crick base pairing, and inhibiting translationthereof. Alternatively, the DNA may be inverted relative to its normalorientation for transcription and so express an RNA transcript that iscomplementary to the target mRNA molecule (i.e., the RNA transcript ofthe anti-sense nucleic acid molecule can hybridize to the target mRNAmolecule through Watson-Crick base pairing). An anti-sense nucleic acidmolecule may be constructed in a number of different ways provided thatit is capable of interfering with the expression of a target protein.Typical anti-sense oligonucleotides to be screened preferably are 30-40nucleotides in length. The anti-sense nucleic acid molecule generallywill be substantially identical (although in antisense orientation) tothe target gene. The minimal identity will typically be greater thanabout 80%, but a higher identity might exert a more effective repressionof expression of the endogenous sequences. Substantially greateridentity of more than about 90% is preferred, though about 95% toabsolute identity would be most preferred.

[0145] Candidate nucleic acid molecules can possess ribozyme activity.Thus, the methods of the invention can be used to screen for ribozymemolecules that inhibit the functional expression of one or more mRNAmolecules that encode one or more proteins that mediate an IL-1 etadependent cellular response. Ribozymes are catalytic RNA molecules thatcan cleave nucleic acid molecules having a sequence that is completelyor partially homologous to the sequence of the ribozyme. It is possibleto design ribozyme transgenes that encode RNA ribozymes thatspecifically pair with a target RNA and cleave the phosphodiesterbackbone at a specific location, thereby functionally inactivating thetarget RNA. In carrying out this cleavage, the ribozyme is not itselfaltered, and is thus capable of recycling and cleaving other molecules.The inclusion of ribozyme sequences within antisense RNAs confersRNA-cleaving activity upon them, thereby increasing the activity of theantisense constructs.

[0146] The design and use of target RNA-specific ribozymes is describedin Haseloff et al. (Nature, 334:585, 1988; see also U.S. Pat. No.5,646,023), both of which publications are incorporated herein byreference. Tabler et al. (Gene 108:175, 1991) have greatly simplifiedthe construction of catalytic RNAs by combining the advantages of theanti-sense RNA and the ribozyme technologies in a single construct.Smaller regions of homology are required for ribozyme catalysis,therefore this can promote the repression of different members of alarge gene family if the cleavage sites are conserved.

[0147] Use of IL-1 eta Polynucleotides and Oligonucleotides

[0148] Among the uses of polynucleotides of the invention is the use offragments as probes or primers. Such fragments generally comprise atleast about 17 contiguous nucleotides of a DNA sequence. In otherembodiments, a DNA fragment comprises at least 30, or at least 60,contiguous nucleotides of a DNA sequence.

[0149] Because homologs of SEQ ID NO:1, from other mammalian species,are contemplated herein, probes based on the human DNA sequence of SEQID NO:1 may be used to screen cDNA libraries derived from othermammalian species, using conventional cross-species hybridizationtechniques.

[0150] Using knowledge of the genetic code in combination with the aminoacid sequences set forth above, sets of degenerate oligonucleotides canbe prepared. Such oligonucleotides are useful as primers, e.g., inpolymerase chain reactions (PCR), whereby DNA fragments are isolated andamplified.

[0151] Polynucleotides encoding SEQ ID NO:2 or oligonucleotide fragmentsof such polynucleotides, can be used by those skilled in the art usingwell-known techniques to identify the human chromosome 2, as well as thespecific locus thereof, that contains the DNA of IL-1 ligand familymembers. Useful techniques include, but are not limited to, usingpolynucleotides or fragments as probes or primers in techniques thatinclude radiation hybrid mapping (high resolution), in situhybridization to chromosome spreads (moderate resolution), and Southernblot hybridization to hybrid cell lines containing individual humanchromosomes (low resolution).

[0152] For example, chromosomes can be mapped by radiation hybridizationwhich can inlude performing PCR amplification using the WhiteheadInstitute/MIT Center for Genome Research Genebridge4 panel of 93radiation hybrids(http://www-genome.wi.mit.edu/ftp/distribution/human_STS_releases/july97/rhmap/genebridge4.html).Useful PCR primers are those that lie within the gene of interest andwhich amplify a product from human genomic DNA, but do not amplifyhamster genomic DNA. The products of the PCR reactions are convertedinto a data vector that is submitted to the Whitehead/MIT RadiationMapping site on the Internet (http://www-seq.wi.mit.edu). The data isscored and the chromosomal assignment and placement relative to knownSequence Tag Site (STS) markers on the radiation hybrid map is provided.The web sitehttp://www-genome.wi.mit.edu/ftp/distribution/human_STS_releases/july97/07-97.INTRO.html)also provides information about radiation hybrid mapping.

[0153] As set forth below, using radiation hybridization, thepolynucleotide of SEQ ID NO:1 is shown to map to the 2q11-12 region ofhuman chromosome 2. Human chromosome 2 is associated with specificdiseases which include but are not limited to glaucoma, ectodermaldysplasia, insulin-dependent diabetes mellitus, wrinkly skin syndrome,T-cell leukemia/lymphoma, and tibial muscular dystrophy. Thus, thepolynucleotide of SEQ ID NO:1 or a fragment thereof can be used by oneskilled in the art using well-known techniques to analyze abnormalitiesassociated with gene mapping to chromosome 2. This enables one todistinguish conditions in which this marker is rearranged or deleted. Inaddition, the polynucleotide of SEQ ID NO:1 or a fragment thereof can beused as a positional marker to map other genes of unknown location.

[0154] DNA of the present invention may be used in developing treatmentsfor any disorder mediated (directly or indirectly) by defective, orinsufficient amounts of, the genes corresponding to the polynucleotidesof the invention. Disclosure herein of native nucleotide sequencespermits the detection of defective genes, and the replacement thereofwith normal genes. Defective genes may be detected in in vitrodiagnostic assays, and by comparison of a native nucleotide sequencedisclosed herein with that of a gene derived from a person suspected ofharboring a defect in this gene.

[0155] Other useful fragments of the polynucleotides of this inventioninclude antisense or sense oligonucleotides comprising a single-strandedpolynucleotide sequence (either RNA or DNA) capable of binding to targetmRNA (sense) or DNA (antisense) sequences. Antisense or senseoligonucleotides according to the present invention comprise a fragmentof DNA (SEQ ID NO:1). Such a fragment generally comprises at least about14 nucleotides, preferably from about 14 to about 30 nucleotides. Theability to derive an antisense or a sense oligonucleotide, based upon acDNA sequence encoding a given protein is described in, for example,Stein and Cohen (Cancer Res. 48:2659, 1988) and van der Krol et al.(BioTechniques 6:958, 1988).

[0156] Binding antisense or sense oligonucleotides to targetpolynucleotide sequences results in the formation of duplexes that blockor inhibit protein expression by one of several means, includingenhanced degradation of the mRNA by RNAseH, inhibition of splicing,premature termination of transcription or translation, or by othermeans. The antisense oligonucleotides thus may be used to blockexpression of proteins. Antisense or sense oligonucleotides furthercomprise oligonucleotides having modified sugar-phosphodiester backbones(or other sugar linkages, such as those described in WO91/06629) andwherein such sugar linkages are resistant to endogenous nucleases. Sucholigonucleotides with resistant sugar linkages are stable in vivo (i.e.,capable of resisting enzymatic degradation) but retain sequencespecificity to be able to bind to target nucleotide sequences.

[0157] Other examples of sense or antisense oligonucleotides includethose oligonucleotides which are covalently linked to organic moieties,such as those described in WO 90/10448, and other moieties thatincreases affinity of the oligonucleotide for a target polynucleotidesequence, such as poly-(L-lysine). Further still, intercalating agents,such as ellipticine, and alkylating agents or metal complexes may beattached to sense or antisense oligonucleotides to modify bindingspecificities of the antisense or sense oligonucleotide for the targetnucleotide sequence.

[0158] Antisense or sense oligonucleotides may be introduced into a cellcontaining the target polynucleotide sequence by any gene transfermethod, including, for example, lipofection, CaPO₄-mediated DNAtransfection, electroporation, or by using gene transfer vectors such asEpstein-Barr virus.

[0159] Sense or antisense oligonucleotides also may be introduced into acell containing the target nucleotide sequence by formation of aconjugate with a ligand binding molecule, as described in WO 91/04753.Suitable ligand binding molecules include, but are not limited to, cellsurface receptors, growth factors, other cytokines, or other ligandsthat bind to cell surface receptors. Preferably, conjugation of theligand binding molecule does not substantially interfere with theability of the ligand binding molecule to bind to its correspondingmolecule or receptor, or block entry of the sense or antisenseoligonucleotide or its conjugated version into the cell.

[0160] Alternatively, a sense or an antisense oligonucleotide may beintroduced into a cell containing the target polynucleotide sequence byformation of an oligonucleotide-lipid complex, as described in WO90/10448. The sense or antisense oligonucleotide-lipid complex ispreferably dissociated within the cell by an endogenous lipase.

[0161] Uses of IL-1 eta Polypeptides and Fragmented Polypeptides

[0162] Polypeptides of the present invention find use as a proteinpurification reagent. The polypeptides may be attached to a solidsupport material and used to purify the binding partner proteins byaffinity chromatography. In particular embodiments, a polypeptide (inany form described herein that is capable of binding the bindingpartner) is attached to a solid support by conventional procedures. Asone example, chromatography columns containing functional groups thatwill react with functional groups on amino acid side chains of proteinsare available (Pharmacia Biotech, Inc., Piscataway, N.J.). In analternative, a polypeptide/Fc protein (as discussed above) is attachedto Protein A- or Protein G-containing chromatography columns throughinteraction with the Fc moiety.

[0163] The polypeptide also finds use in purifying or identifying cellsthat express the binding partner on the cell surface. Polypeptides arebound to a solid phase such as a column chromatography matrix or asimilar suitable substrate. For example, magnetic microspheres can becoated with the polypeptides and held in an incubation vessel through amagnetic field. Suspensions of cell mixtures containing the bindingpartner expressing cells are contacted with the solid phase having thepolypeptides thereon. Cells expressing the binding partner on the cellsurface bind to the fixed polypeptides, and unbound cells then arewashed away.

[0164] Alternatively, the polypeptides can be conjugated to a detectablemoiety, then incubated with cells to be tested for binding partnerexpression. After incubation, unbound labeled matter is removed and thepresence or absence of the detectable moiety on the cells is determined.

[0165] In a further alternative, mixtures of cells suspected ofcontaining cells expressing the binding partner are incubated withbiotinylated polypeptides. Incubation periods are typically at least onehour in duration to ensure sufficient binding. The resulting mixturethen is passed through a column packed with avidin-coated beads, wherebythe high affinity of biotin for avidin provides binding of the desiredcells to the beads. Procedures for using avidin-coated beads are known(see Berenson, et al. J. Cell. Biochem., 10D:239, 1986). Washing toremove unbound material, and the release of the bound cells, areperformed using conventional methods.

[0166] Polypeptides also find use in measuring the biological activityof the binding partner protein in terms of their binding affinity. Thepolypeptides thus may be employed by those conducting “qualityassurance” studies, e.g., to monitor shelf life and stability of proteinunder different conditions. For example, the polypeptides may beemployed in a binding affinity study to measure the biological activityof a binding partner protein that has been stored at differenttemperatures, or produced in different cell types. The proteins also maybe used to determine whether biological activity is retained aftermodification of a binding partner protein (e.g., chemical modification,truncation, mutation, etc.). The binding affinity of the modifiedbinding partner protein is compared to that of an unmodified bindingpartner protein to detect any adverse impact of the modifications onbiological activity of the binding partner. The biological activity of abinding partner protein thus can be ascertained before it is used in aresearch study, for example.

[0167] The polypeptides also find use as carriers for delivering agentsattached thereto to cells bearing the binding partner. The polypeptidesthus can be used to deliver diagnostic or therapeutic agents to suchcells (or to other cell types found to express the binding partner onthe cell surface) in in vitro or in vivo procedures.

[0168] Detectable (diagnostic) and therapeutic agents that may beattached to a polypeptide include, but are not limited to, toxins, othercytotoxic agents, drugs, radionuclides, chromophores, enzymes thatcatalyze a colorimetric or fluorometric reaction, and the like, with theparticular agent being chosen according to the intended application.Among the toxins are ricin, abrin, diphtheria toxin, Pseudomonasaeruginosa exotoxin A, ribosomal inactivating proteins, mycotoxins suchas trichothecenes, and derivatives and fragments (e.g., single chains)thereof. Radionuclides suitable for diagnostic use include, but are notlimited to, ¹²³I, ¹³¹I, ^(99m)Tc, ¹¹¹In, and ⁷⁶Br. Examples ofradionuclides suitable for therapeutic use are ¹³¹I, ²¹¹ At, ⁷⁷Br,¹⁸⁶Re, ¹⁸⁸Re, ²¹²Pb, ²¹²Bi, ¹⁰⁹Pd, ⁶⁴Cu, and ⁶1Cu.

[0169] Such agents may be attached to the polypeptide by any suitableconventional procedure. The polypeptide comprises functional groups onamino acid side chains that can be reacted with functional groups on adesired agent to form covalent bonds, for example. Alternatively, theprotein or agent may be derivatized to generate or attach a desiredreactive functional group. The derivatization may involve attachment ofone of the bifunctional coupling reagents available for attachingvarious molecules to proteins (Pierce Chemical Company, Rockford, Ill.).A number of techniques for radiolabeling proteins are known.Radionuclide metals may be attached to polypeptides by using a suitablebifunctional chelating agent, for example.

[0170] Conjugates comprising polypeptides and a suitable diagnostic ortherapeutic agent (preferably covalently linked) are thus prepared. Theconjugates are administered or otherwise employed in an amountappropriate for the particular application.

[0171] Polypeptides of the invention may be used in developingtreatments for any disorder mediated (directly or indirectly) bydefective, or insufficient amounts of the polypeptides. Further, thepolypeptides of the invention may be used in developing treatments forany disorder resulting (directly or indirectly) from an excess of thepolypeptide. The polypeptides of the instant invention may beadministered to a mammal afflicted with such disorders.

[0172] The polypeptides may also be employed in inhibiting a biologicalactivity of the binding partner, in in vitro or in vivo procedures. Forexample, a purified IL-1 eta polypeptide can be used to inhibit bindingof endogenous IL-1 eta to its cell surface receptor.

[0173] Polypeptides of the invention may be administered to a mammal totreat a binding partner-mediated disorder. Such binding partner-mediateddisorders include conditions caused (directly or indirectly) orexacerbated by the binding partner.

[0174] Compositions of the present invention may contain a polypeptidein any form described herein, such as native proteins, variants,derivatives, oligomers, and biologically active fragments. In particularembodiments, the composition comprises a soluble polypeptide or anoligomer comprising soluble polypeptides of the invention.

[0175] Compositions comprising an effective amount of a polypeptide ofthe present invention, in combination with other components such as aphysiologically acceptable diluent, carrier, or excipient, are providedherein. The polypeptides can be formulated according to known methodsused to prepare pharmaceutically useful compositions. They can becombined in admixture, either as the sole active material or with otherknown active materials suitable for a given indication, withpharmaceutically acceptable diluents (e.g., saline, Tris-HCl, acetate,and phosphate buffered solutions), preservatives (e.g., thimerosal,benzyl alcohol, parabens), emulsifiers, solubilizers, adjuvants and/orcarriers. Suitable formulations for pharmaceutical compositions includethose described in Remington's Pharmaceutical Sciences, 16th ed. 1980,Mack Publishing Company, Easton, Pa.

[0176] In addition, such compositions can be complexed with polyethyleneglycol (PEG), metal ions, or incorporated into polymeric compounds suchas polyacetic acid, polyglycolic acid, hydrogels, dextran, etc., orincorporated into liposomes, microemulsions, micelles, unilamellar ormultilamellar vesicles, erythrocyte ghosts or spheroblasts. Suchcompositions will influence the physical state, solubility, stability,rate of in vivo release, and rate of in vivo clearance, and are thuschosen according to the intended application.

[0177] The compositions of the invention can be administered in anysuitable manner, e.g., topically, parenterally, or by inhalation. Theterm “parenteral” includes injection, e.g., by subcutaneous,intravenous, or intramuscular routes, also including localizedadministration, e.g., at a site of disease or injury. Those of ordinaryskill in the art recognize that other types of localized administration(e.g., intraarticular, intracapsular, intracarpal, intracelial,intracerebroventricular, intrasynovial, intraspinal, intraligamentus,intrameningeal, intraocular, epidural, transepithelially, and/oradministration by one or more of these routes at a site near or adjacentto a site of disease or injury) are suitable for use in administeringthe compositions of the present invention. Sustained release fromimplants is also contemplated.

[0178] One skilled in the pertinent art will recognize that suitabledosages will vary, depending upon such factors as the nature of thedisorder to be treated, the patient's body weight, age, and generalcondition, and the route of administration. Preliminary doses can bedetermined according to animal tests, and the scaling of dosages forhuman administration is performed according to art-accepted practices.

[0179] Compositions comprising polynucleotides in physiologicallyacceptable formulations are also contemplated. DNA may be formulated forinjection, for example. Moreover, inasmuch as those of ordinary skill inthe art are aware that nucleic acid compositions (including DNA) aretaken up by cells and result in the expression of protein in or near thearea where the nucleic acid composition was administered, the inventivenucleic acid compositions will be useful for localized administration ofpolypeptides encoded thereby.

[0180] Another use of the polypeptide of the present invention is as aresearch tool for studying the biological effects that result from theinteractions of IL-1 eta with its binding partner, or from inhibitingthese interactions, on different cell types. Polypeptides also may beemployed in it vitro assays for detecting IL-1 eta, the binding partneror the interaction thereof. The inventive polypeptides will also beuseful in elucidating the signaling pathways of IL-1 family members, andin identifying molecules that modulate various aspects of such signalingpathways. The modulators identified by studies utilizing the inventivepolypeptides have utility in treating or ameliorating a wide variety ofdiseases and syndromes in which the inflammatory response plays a role.

[0181] Another embodiment of the invention relates to uses of thepolypeptides of the invention to study cell signal transduction. IL-1family ligands play a central role in protection against infection andimmune inflammatory responses which includes cellular signaltransduction, activating vascular endothelial cells and lymphocytes,induction of inflammatory cytokines, acute phase proteins,hematopoiesis, fever, bone resorption, prostaglandins,metalloproteinases, and adhesion molecules. With the continued increasein the number of known IL-1 family members, a suitable classificationscheme is one based on comparing polypeptide structure as well asfunction (activation and regulatory properties). Thus, IL-1 eta, likeother IL-1 family ligands (IL-1α, IL-1β, and IL-18) are likely involvedin many of the functions noted above as well as promote inflammatoryresponses and therefore be involved in the causation and maintenance ofinflammatory and/or autoimmune diseases such as rheumatoid arthritis,inflammatory bowel disease, and psoriasis. As such, alterations in theexpression and/or activation of the polypeptides of the invention canhave profound effects on a plethora of cellular processes, including,but not limited to, activation or inhibition of cell specific responsesand proliferation. Expression of cloned IL-1 eta, or of functionallyinactive mutants thereof, can be used to identify the role a particularprotein plays in mediating specific signaling events.

[0182] Accordingly, IL-1 eta has therapeutic uses, such as protectingagainst infection and generating immune and inflammatory responses inindividuals whose immune and inflammatory responses are inappropriate ornonresponsive. For example, IL-1 eta may be useful in stimulating theimmune system of individuals whose immune system is immunosuppressed.Similarly, because IL-1 eta likely promotes inflammatory responses andis involved in the causation and maintenance of inflammatory and/orautoimmune diseases, antagonists of IL-1 eta are useful in inhibiting ortreating inflammatory and/or automimmune disease. Thus, antagonists ofIL-1 eta will be useful in treating inflammatory bowel disease (forexample, Crohn's disease and ulcerative colitis), multiple sclerosis(MS) and other demyelinating conditions, and asthma or other pulmonaryconditions in which an immune or inflammatory response is involved (forexample, infection-associated airway hyperactivity, granulomatous lungdisease, emphysema and chronic fibrosing alveolitis and acute hyperoxiclung damage).

[0183] IL-1 mediated cellular signaling often involves a molecularactivation cascade, during which a receptor propagates a ligand-receptormediated signal by specifically activating intracellular kinases whichphosphorylate target substrates. These substrates can themselves bekinases which become activated following phosphorylation. Alternatively,they can be adaptor molecules that facilitate down stream signalingthrough protein-protein interaction following phosphorylation.Regardless of the nature of the substrate molecule(s), expressedfunctionally active versions of IL-1 eta and its binding partners can beused to identify what substrate(s) were recognized and activated by thepolypeptides of the invention. As such, these novel polypeptides can beused as reagents to identify novel molecules involved in signaltransduction pathways.

[0184] Moreover, as described herein, IL-1 eta can be used to identifyantagonists of such signaling pathways. Therefore, administration ofIL-1 eta antagonists will have therapeutic application in blockinginflammatory responses, including the activation of transcriptionfactors NFkappaB and API, the protein kinases Jun N-terminal kinase andp38 MAP kinase, the enzymes COX-2 leading to prostaglandin productionand iNOS leading to nitric oxide production, and inflammation ingeneral. Such signaling pathways have been shown to be involved insepsis, septic, toxic or hemorhagic shock and acute respiratorydistress, such as that which occurs in inhalational anthrax. Antagonistsof IL-1 eta can be used in combination with other agents in thetreatment of inflammatory dysregulation syndromes, including for exampleinhibitors of TNFalpha, inhibitors of other members of the IL-1 family,corticosteroids, and inhibitors of other mediators of inflammation suchas macrophage migration inhibitory factor, and/or inhibitors ofcell-surface receptors such as CD14 and Toll-like receptors.

[0185] Similarly, because IL-1 promotes inflammatory responses and isinvolved in the causation and maintenance of inflammatory and/orautoimmune diseases, antagonists of IL-1 eta are useful in inhibiting ortreating inflammatory and/or autoimmune disease. Thus, IL-1 etaantagonists will be useful in treating arthritic conditions that have aninflammatory or autoimmune component, for example, rheumatoid arthritisand/or ankylosing spondylitis; inflammatory bowel disease, includingCrohn's Disease and ulcerative colitis, and psoriasis (includingpsoriatic arthritis). Other inflammatory and/or autoimmune diseases inwhich IL-1 eta is implicated include pulmonary conditions relating to animmune or inflammatory response and/or in which airway hyperreactivityplays a role, for example, asthma, infection-associated airwayhyperactivity, granulomatous lung disease, emphysema and chronicfibrosing alveolitis and acute hyperoxic lung damage, and demyelinatingconditions that have an inflammatory or autoimmune component, forexample, multiple sclerosis and/or chronic inflammatory demyelinatingpolyneuropathy. Accordingly, antagonists of IL-1 eta will also be usefulin ameliorating these conditions.

[0186] Additional conditions for which an autoimmune and/or inflammatorycomponent is a contributory factor (and thus, for which antagonists ofIL-1 eta are useful) include cardiovascular conditions such as stroke,acute myocardial infarction, unstable angina, arterial restenosis andcongestive heart failure. IL-1 eta antagonists are useful in treating orpreventing osteoporosis and/or osteoarthritis, as well asglomerulonephritis, uveitis, and/or Behçet's syndrome. An autoimmune orinflammatory component also plays a role in the cause or maintenance ofsepsis, acute pancreatitis, diabetes (particularly Type II, insulindependent diabetes), endometriosis, and periodontal disease. Similarly,the inflammatory response causes or exacerbates heat stroke andglaucoma, and the cytokines involved in the immune/inflammatory responseplay a supportive role in neoplastic disease (for example, in multiplemyeloma and/or myeloid leukemia), facilitating the growth of neoplasticcells. Accordingly, IL-1 eta antagonists are useful in treating orameliorating these conditions by downregulating the immune and/orinflammatory response that plays a causative role therein.

[0187] Moreover, as disclosed in United States Patent Application20010026801 A1, published Oct. 4, 2001, other syndromes and/orconditions are caused or exacerbated by localized production ofproinflammatory cytokines. Accordingly, antagonists of IL-1 eta can beadministered locally to ameliorate a localized inflammatory and/orautoimmune reaction. Such localized reactions occur, for example, inneurological disorders due to a herniated nucleus pulposus (herniateddisk), osteoarthritis, other forms of arthritis, disorders of bone,disease, and/or trauma causing damage to the optic nerve, other cranialnerves, spinal cord, nerve roots, or peripheral nerves. Moreover,trauma, injury, compression and disease can affect individual nerves,nerve roots, the spinal cord, or localized areas of muscle. Disordersfor which localized administration of antagonists of IL-1 are usefulinclude spinal cord injury, spinal cord compression, spinal stenosis,carpal tunnel syndrome, glaucoma, Bell's palsy, localized musculardisorders (including acute muscle pulls, muscle sprains, muscle tears,and muscle spasm), Alzheimer's disease and post-herpetic neuralgia.Localized anti-inflammatory agents will also be useful for treatment ofconditions in which fascia, tendons, ligaments or other structures of ajoint, and/or other connective tissues are injured and/or inflamed (forexample, tendonitis, bursitis, strained, sprained or torn ligaments,fascitis, etc.). Useful antagonists for localized administration in theaforementioned conditions includes localized administration ofpolypeptide compositions as well as nucleic acid compositions, aspreviously described herein.

[0188] Antibodies

[0189] Antibodies that are immunoreactive with the polypeptides of theinvention are provided herein. Such antibodies specifically bind to thepolypeptides via the antigen-binding sites of the antibody (as opposedto non-specific binding). Thus, the polypeptides, fragments, variants,fusion proteins, etc., as set forth above may be employed as“immunogens” in producing antibodies immunoreactive therewith. Morespecifically, the polypeptides, fragment, variants, fusion proteins,etc. contain antigenic determinants or epitopes that elicit theformation of antibodies.

[0190] These antigenic determinants or epitopes can be either linear orconformational (discontinuous). Linear epitopes are composed of a singlesection of amino acids of the polypeptide, while conformational ordiscontinuous epitopes are composed of amino acids sections fromdifferent regions of the polypeptide chain that are brought into closeproximity upon protein folding (C. A. Janeway, Jr. and P. Travers,Immuno Biology 3:9 (Garland Publishing Inc., 2nd ed. 1996)). Becausefolded proteins have complex surfaces, the number of epitopes availableis quite numerous; however, due to the conformation of the protein andsteric hinderances, the number of antibodies that actually bind to theepitopes is less than the number of available epitopes (C. A. Janeway,Jr. and P. Travers, Immuno Biology 2:14 (Garland Publishing Inc., 2nded. 1996)). Epitopes may be identified by any of the methods known inthe art.

[0191] Thus, one aspect of the present invention relates to theantigenic epitopes of the polypeptides of the invention. Such epitopesare useful for raising antibodies, in particular monoclonal antibodies,as described in more detail below. Additionally, epitopes from thepolypeptides of the invention can be used as research reagents, inassays, and to purify specific binding antibodies from substances suchas polyclonal sera or supernatants from cultured hybridomas. Suchepitopes or variants thereof can be produced using techniques well knownin the art such as solid-phase synthesis, chemical or enzymatic cleavageof a polypeptide, or using recombinant DNA technology.

[0192] As to the antibodies that can be elicited by the epitopes of thepolypeptides of the invention, whether the epitopes have been isolatedor remain part of the polypeptides, both polyclonal and monoclonalantibodies may be prepared by conventional techniques. See, for example,Monoclonal Antibodies, Hybridomas: A New Dimension in BiologicalAnalyses, Kennet et al. (eds.), Plenum Press, New York (1980); andAntibodies: A Laboratory Manual, Harlow and Land (eds.), Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1988).

[0193] Hybridoma cell lines that produce monoclonal antibodies specificfor the polypeptides of the invention are also contemplated herein. Suchhybridomas may be produced and identified by conventional techniques.One method for producing such a hybridoma cell line comprises immunizingan animal with a polypeptide; harvesting spleen cells from the immunizedanimal; fusing said spleen cells to a myeloma cell line, therebygenerating hybridoma cells; and identifying a hybridoma cell line thatproduces a monoclonal antibody that binds the polypeptide. Themonoclonal antibodies may be recovered by conventional techniques.

[0194] The monoclonal antibodies of the present invention includechimeric antibodies, e.g., humanized versions of murine monoclonalantibodies. Such humanized antibodies may be prepared by knowntechniques and offer the advantage of reduced immunogenicity when theantibodies are administered to humans. In one embodiment, a humanizedmonoclonal antibody comprises the variable region of a murine antibody(or just the antigen binding site thereof) and a constant region derivedfrom a human antibody. Alternatively, a humanized antibody fragment maycomprise the antigen binding site of a murine monoclonal antibody and avariable region fragment (lacking the antigen-binding site) derived froma human antibody. Procedures for the production of chimeric and furtherengineered monoclonal antibodies include those described in Riechmann etal. (Nature 332:323, 1988), Liu et al. (PNAS 84:3439, 1987), Larrick etal. (Bio/Technology 7:934, 1989), and Winter and Harris (TIPS 14:139,May, 1993). Procedures to generate antibodies transgenically can befound in GB 2,272,440, U.S. Pat. Nos. 5,569,825 and 5,545,806 andrelated patents claiming priority therefrom, all of which areincorporated by reference herein.

[0195] Antigen-binding fragments of the antibodies, which may beproduced by conventional techniques, are also encompassed by the presentinvention. Examples of such fragments include, but are not limited to,Fab and F(ab′)₂ fragments. Antibody fragments and derivatives producedby genetic engineering techniques are also provided.

[0196] In one embodiment, the antibodies are specific for thepolypeptides of the present invention and do not cross-react with otherproteins. Screening procedures by which such antibodies may beidentified are well known, and may involve immunoaffinitychromatography, for example.

[0197] Uses Thereof

[0198] The antibodies of the invention can be used in assays to detectthe presence of the polypeptides or fragments of the invention, eitherin vitro or in vivo. The antibodies also may be employed in purifyingpolypeptides or fragments of the invention by immunoaffinitychromatography.

[0199] Those antibodies that additionally can block binding of thepolypeptides of the invention to the binding partner may be used toinhibit a biological activity that results from such binding. Suchblocking antibodies may be identified using any suitable assayprocedure, such as by testing antibodies for the ability to inhibitbinding of IL-1 eta to certain cells expressing the IL-1 eta receptors.Alternatively, blocking antibodies may be identified in assays for theability to inhibit a biological effect that results from polypeptides ofthe invention binding to their binding partners to target cells.Antibodies may be assayed for the ability to inhibit IL-1 eta-mediated,or binding partner-mediated cell lysis, for example.

[0200] Such an antibody may be employed in an in vitro procedure, oradministered in vivo to inhibit a biological activity mediated by theentity that generated the antibody. Disorders caused or exacerbated(directly or indirectly) by the interaction of the polypeptides of theinvention with the binding partner thus may be treated. A therapeuticmethod involves in vivo administration of a blocking antibody to amammal in an amount effective in inhibiting a binding partner-mediatedbiological activity. Monoclonal antibodies are generally preferred foruse in such therapeutic methods. In one embodiment, an antigen-bindingantibody fragment is employed.

[0201] Antibodies may be screened for agonistic (i.e., ligand-mimicking)properties. Such antibodies, upon binding to cell surface receptor,induce biological effects (e.g., transduction of biological signals)similar to the biological effects induced when IL-1 binds to cellsurface IL-1 receptors. Agonistic antibodies may be used to activatevascular endothelial cells and lymphocytes, induce local tissuedestruction and fever (Janeway et al., 1996), stimulate macrophages andvascular endothelial cells to produce IL-6, and upregulate molecules onthe surface of vascular endothelial cells.

[0202] Compositions comprising an antibody that is directed againstpolypeptides of the invention, and a physiologically acceptable diluent,excipient, or carrier, are provided herein. Suitable components of suchcompositions are as described above for compositions containingpolypeptides of the invention.

[0203] Also provided herein are conjugates comprising a detectable(e.g., diagnostic) or therapeutic agent, attached to the antibody.Examples of such agents are presented above. The conjugates find use inin vitro or in vivo procedures.

[0204] The following examples are offered by way of illustration, andnot by way of limitation. Those skilled in the art will recognize thatvariations of the invention embodied in the examples can be made,especially in light of the teachings of the various references citedherein, the disclosures of which are incorporated by reference in theirentirety.

EXAMPLE 1 Isolation of the IL-1 eta Polynucleotides

[0205] Human genomic DNA containing the upstream portion of IL-1 etacDNA as defined in EP 0879889A2 was cloned and extended in the 3′direction. The genomic DNA was sequenced and examined for potentialhomology to the C-terminal section of IL-1 family members. A region withthe potential to code with homology to the C-terminal section of IL-1family members was located and is disclosed as polynucleotides 375 to585 of SEQ. ID. NO.:1. PCR primers were synthesized containing the stopcodon in the 3′ or reverse primer, and the initiating ATG of the IL-1eta cDNA (SEQ. ID. NO.:1 of EP 0879889A2) in the 5′ or sense primer.Using these primers, IL-1 eta cDNA was amplified from first stand cDNAmade from human tonsil mRNA. PCR was preformed using standard protocols.

EXAMPLE 2 Use of Purified IL-1 eta Polypeptides

[0206] Serial dilutions of IL-1 eta-containing samples (in 50 mM NaHCO₃,brought to pH 9 with NaOH) are coated onto Linbro/Fitertek 96 well flatbottom E.I.A. microtitration plates (ICN Biomedicals Inc., Aurora, Ohio)at 100:1/well. After incubation at 4° C. for 16 hours, the wells arewashed six times with 200:1 PBS containing 0.05% Tween-20 (PBS-Tween).The wells are then incubated with FLAG®-binding partner at 1 mg/ml inPBS-Tween with 5% fetal calf serum (FCS) for 90 minutes (100:1 perwell), followed by washing as above. Next, each well is incubated withthe anti-FLAG® (monoclonal antibody M2 at 1 mg/ml in PBS-Tweencontaining 5% FCS for 90 minutes (100:1 per well), followed by washingas above. Subsequently, wells are incubated with a polyclonal goatanti-mIgG1-specific horseradish peroxidase-conjugated antibody (a 1:5000dilution of the commercial stock in PBS-Tween containing 5% FCS) for 90minutes (100:1 per well). The HRP-conjugated antibody is obtained fromSouthern Biotechnology Associates, Inc., Birmingham, Ala. Wells then arewashed six times, as above.

[0207] For development of the ELISA, a substrate mix [100:1 per well ofa 1:1 premix of the TMB Peroxidase Substrate and Peroxidase Solution B(Kirkegaard Perry Laboratories, Gaithersburg, Md.)] is added to thewells. After sufficient color reaction, the enzymatic reaction isterminated by addition of 2 N H₂SO₄ (50:1 per well). Color intensity(indicating ligand receptor binding) is determined by measuringextinction at 450 nm on a V Max plate reader (Molecular Devices,Sunnyvale, Calif.).

EXAMPLE 3 Amino Acid Sequence

[0208] The amino acid sequence of IL-1 eta was determined by translationof the nucleotide sequence of SEQ ID NO:1. The coding region includesnucleotide residues 112-585.

EXAMPLE 4 DNA and Amino Acid Sequences

[0209] The nucleotide sequence of the isolated IL-1 eta and the aminoacid sequence encoded thereby, are presented in SEQ ID NOs:1 and 2. Thesequence of the IL-1 eta DNA fragment isolated by PCR corresponds tonucleotides 1 to 585 of SEQ ID NO:1. Nucleotide residues 112-585 encodeamino acids 1 to 157 of SEQ ID NO:2.

[0210] The amino acid sequence of SEQ ID NO:2 bears significant homologyto other known IL-1 ligand family members.

EXAMPLE 5 Monoclonal Antibodies that Bind Polypeptides of the Invention

[0211] This example illustrates a method for preparing monoclonalantibodies that bind IL-1 eta. Suitable immunogens that may be employedin generating such antibodies include, but are not limited to, purifiedIL-1 eta polypeptide or an immunogenic fragment thereof such as theextracellular domain, or fusion proteins containing IL-1 eta (e.g., asoluble IL-1 eta/Fc fusion protein).

[0212] Purified IL-1 eta can be used to generate monoclonal antibodiesimmunoreactive therewith, using conventional techniques such as thosedescribed in U.S. Pat. No. 4,411,993. Briefly, mice are immunized withIL-1 eta immunogen emulsified in complete Freund's adjuvant, andinjected in amounts ranging from 10-100 g subcutaneously orintraperitoneally. Ten to twelve days later, the immunized animals areboosted with additional IL-1 eta emulsified in incomplete Freund'sadjuvant. Mice are periodically boosted thereafter on a weekly tobi-weekly immunization schedule. Serum samples are periodically taken byretro-orbital bleeding or tail-tip excision to test for IL-1 etaantibodies by dot blot assay, ELISA (Enzyme-Linked Immunosorbent Assay)or inhibition of IL-1 eta receptor binding.

[0213] Following detection of an appropriate antibody titer, positiveanimals are provided one last intravenous injection of IL-1 eta insaline. Three to four days later, the animals are sacrificed, spleencells harvested, and spleen cells are fused to a murine myeloma cellline, e.g., NS1 or preferably P3x63Ag8.653 (ATCC CRL 1580). Fusionsgenerate hybridoma cells, which are plated in multiple microtiter platesin a HAT (hypoxanthine, aminopterin and thymidine) selective medium toinhibit proliferation of non-fused cells, myeloma hybrids, and spleencell hybrids.

[0214] The hybridoma cells are screened by ELISA for reactivity againstpurified IL-1 eta by adaptations of the techniques disclosed in Engvallet al., (Immunochem. 8:871, 1971) and in U.S. Pat. No. 4,703,004. Apreferred screening technique is the antibody capture techniquedescribed in Beckmann et al., (J. Immunol. 144:4212, 1990). Positivehybridoma cells can be injected intraperitoneally into syngeneic BALB/cmice to produce ascites containing high concentrations of anti-IL-1 etamonoclonal antibodies. Alternatively, hybridoma cells can be grown invitro in flasks or roller bottles by various techniques. Monoclonalantibodies produced in mouse ascites can be purified by ammonium sulfateprecipitation, followed by gel exclusion chromatography. Alternatively,affinity chromatography based upon binding of antibody to Protein A orProtein G can also be used, as can affinity chromatography based uponbinding to IL-1 eta.

EXAMPLE 6 Northern Blot Analysis

[0215] The tissue distribution of IL-1 eta is investigated by Northernblot analysis, as follows. An aliquot of a radiolabeled riboprobe isadded to two different human multiple tissue Northern blots (Clontech,Palo Alto, Calif.; Biochain, Palo Alto, Calif.). The blots arehybridized in 10× Denhardts, 50 mM Tris pH 7.5, 900 mM NaCl, 0.1% Napyrophosphate, 1% SDS, 200 g/mL salmon sperm DNA. Hybridization isconducted overnight at 63° C. in 50% formamide as previously described(March et al., Nature 315:641-647, 1985). The blots are then washed with2×SSC, 0.1% SDS at 68° C. for 30 minutes. The cells and tissues with thehighest levels of IL-1 eta mRNA are determined by comparison to controlprobing with a β-actin-specific probe.

[0216] Expression of IL-eta was also analyzed in several animal modelsof human disease by conventional real-time polymerase chain reaction(RT-PCR) substantially as described in U.S. Ser. No. 09/876,790, filedJun. 6, 2001, and/or by TaqMan® RT-PCR (Applied Biosystems, Foster City,Calif.).). Total RNA from small or large intestine (colitis models:DSS-induced colitis, anti-CD-3 induced ileitis and MdrKO spontaneouscolitis), spinal cord (multiple sclerosis [MS] models: EAE using SJLmice injected with PLP), or lung (asthma model: BALB/c/OVA-inducedasthma model) was used to make first strand cDNA. The level ofexpression was subjectively scored as a function of relative ethidiumbromide staining intensity.

[0217] Results of these experiments indicated that expression of IL-1eta was upregulated in DSS-induced colitis. Accordingly, IL-1 eta isimplicated in the cause or prolongation of inflammatory bowel disease,and antagonists thereof will be useful in treating or amelioratinginflammatory bowel disease in individuals afflicted with suchconditions. Additionally, IL-1 eta appeared to be upregulated in theearly stages of EAE, indicating that an antagonist thereof may be usefulin treating or ameliorating MS and other demyelinating conditions. IL-1eta was also upregulated in the OVA-induced asthma model, indicatingthat an antagonist thereof may be useful in treating or amelioratingasthma and other pulmonary conditions relating to an immune orinflammatory response.

EXAMPLE 7 Binding Assay

[0218] Full length IL-1 eta can be expressed and tested for the abilityto bind IL-1 eta receptors. The binding assay can be conducted asfollows.

[0219] A fusion protein comprising a leucine zipper peptide fused to theN-terminus of a soluble IL-1 eta polypeptide (LZ-IL-1 eta) is employedin the assay. An expression construct is prepared, essentially asdescribed for preparation of the FLAG® (IL-1 eta) expression constructin Wiley et al. (Immunity, 3:673-682, 1995; hereby incorporated byreference), except that DNA encoding the FLAG® peptide was replaced witha sequence encoding a modified leucine zipper that allows fortrimerization. The construct, in expression vector pDC409, encodes aleader sequence derived from human cytomegalovirus, followed by theleucine zipper moiety fused to the N-terminus of a soluble IL-1 etapolypeptide. The LZ-IL-1 eta is expressed in CHO cells, and purifiedfrom the culture supernatant.

[0220] The expression vector designated pDC409 is a mammalian expressionvector derived from the pDC406 vector described in McMahan et al. (EMBOJ. 10:2821-2832, 1991; hereby incorporated by reference). Features addedto pDC409 (compared to pDC406) include additional unique restrictionsites in the multiple cloning site (mcs); three stop codons (one in eachreading frame) positioned downstream of the mcs; and a T7 polymerasepromoter, downstream of the mcs, that facilitates sequencing of DNAinserted into the mcs.

[0221] For expression of full length human IL-1 eta protein, the entirecoding region (i.e., the DNA sequence presented in SEQ ID NO:1) isamplified by polymerase chain reaction (PCR). The template employed inthe PCR is the cDNA clone isolated from tonsil first strand cDNA, asdescribed in example 1. The isolated and amplified DNA is inserted intothe expression vector pDC409, to yield a construct designatedpDC409-IL-1 eta.

[0222] LZ-IL-1 eta polypeptide is employed to test the ability to bindto host cells expressing recombinant or endogenous IL-1 eta receptors,as discussed above. Cells expressing IL-1 eta receptor are cultured inDMEM supplemented with 10% fetal bovine serum, penicillin, streptomycin,and glutamine. Cells are incubated with LZ-IL-1 eta (5 mg/ml) for about1 hour. Following incubation, the cells are washed to remove unboundLZ-IL-1 eta and incubated with a biotinylated anti-LZ monoclonalantibody (5 mg/ml), and phycoerythrin-conjugated streptavidin (1:400),before analysis by fluorescence-activated cell scanning (FACS). Thecytometric analysis was conducted on a FACscan (Beckton Dickinson, SanJose, Calif.).

[0223] The cells expressing IL-1 eta receptors showed significantlyenhanced binding of LZ-IL-1 eta, compared to the control cells notexpressing IL-1 eta receptors.

EXAMPLE 8 Expression Analysis

[0224] First strand cDNAs present in Clontech (Palo Alto, Calif.) HumanMultiple Tissue cDNA Panels I (Cat. # K1420-1) and II (Cat. #K1421-1)and the Human Immune Panel (Cat. #K1426-1) were screened by PCRamplification using primers (sense: ACATCATGAACCCACAACGGGAGGCAGCAC;antisense: CTCTATCCTGGAACCAGCCACCCACAGC). The primers were designed tospan introns so that products arising from genomic DNA and cDNA could bedistinguished. In some cases, nested primers (sense:CCAAATCCTATGCTATTCGTGATTCTCGAC; antisense:GGATTTATTCCACAGAATCTAAGTAGAAG) were used in a second PCR reaction. Thepresence of an amplification product for each gene/tissue combinationwas determined by analysis on agarose gels stained with ethidiumbromide.

[0225] Alternatively, individual cell types from human peripheral bloodwere isolated and stimulations were performed (Kubin et al., Blood83(7):1847-55 (1994); Kubin et al., J Exp Med 180(1):211-22 (1994)). NKcells were incubated with IL-12 (R&D Biosystems; 1 ng/ml) for either 2hours or 4 hours. T cells were unstimulated or stimulated with anti-CD3(OKT-3 antibody, immobilized on plastic at 5 ng/ml) or with thecombination of anti-CD3 and anti-CD28 (the anti-CD28 antibody was CD248used in soluble form as a 1:500 dilution of ascites fluid), for 30minutes or 4 hours. Monocytes were unstimulated, or stimulated with LPS(Sigma; lug/ml) for 2 or 3 hours. B cells were unstimulated, orstimulated with the combination of 0.05% SAC and 500 ng/ml CD40L trimer(Immunex) and 5 ng/ml IL-4 (Immunex) for 3.5 or 4 hours. Dendritic cellswere stimulated with LPS as for monocytes, for 2 or 4 hours. Afterisolation of RNA and synthesis of first strand cDNA, PCR amplificationsand gel analysis were performed.

[0226] Table I demonstrates the expression of IL-1 eta in lymphoidorgans. A “-” indicates that the mRNA was looked for but not found.Positive results derived by PCR analysis for a panel of first strandcDNAs (Clontech) are designated by an “A”.

EXAMPLE 9 Binding Assay

[0227] This example describes a type of binding assay utilizing theinventive proteins. A recombinant expression vector containing thebinding partner cDNA is constructed using methods well known in the art.CV1-EBNA-1 cells in 10 cm² dishes are transfected with the recombinantexpression vector. CV-1/EBNA-1 cells (ATCC CRL 10478) constitutivelyexpress EBV nuclear antigen-1 driven from the CMV immediate-earlyenhancer/promoter. CV1-EBNA-1 was derived from the African Green Monkeykidney cell line CV-1 (ATCC CCL 70), as described by McMahan et al.(EMBO J. 10:2821, 1991).

[0228] The transfected cells are cultured for 24 hours, and the cells ineach dish then are split into a 24-well plate. After culturing anadditional 48 hours, the transfected cells (about 4×10⁴ cells/well) arewashed with BM-NFDM, which is binding medium (RPMI 1640 containing 25mg/ml bovine serum albumin, 2 mg/ml sodium azide, 20 mM Hepes pH 7.2) towhich 50 mg/ml nonfat dry milk has been added. The cells then areincubated for 1 hour at 37° C. with various concentrations of, forexample, a soluble polypeptide/Fc fusion protein made as set forthabove. Cells then are washed and incubated with a constant saturatingconcentration of a ¹²⁵I-mouse anti-human IgG in binding medium, withgentle agitation for 1 hour at 37° C. After extensive washing, cells arereleased via trypsinization.

[0229] The mouse anti-human IgG employed above is directed against theFc region of human IgG and can be obtained from Jackson ImmunoresearchLaboratories, Inc., West Grove, Pa. The antibody is radioiodinated usingthe standard chloramine-T method. The antibody will bind to the Fcportion of any polypeptide/Fc protein that has bound to the cells. Inall assays, non-specific binding of ¹²⁵I-antibody is assayed in theabsence of the Fc fusion protein/Fc, as well as in the presence of theFc fusion protein and a 200-fold molar excess of unlabeled mouseanti-human IgG antibody.

[0230] Cell-bound ¹²⁵I-antibody is quantified on a Packard Autogammacounter. Affinity calculations (Scatchard, Ann. N.Y. Acad. Sci. 51:660,1949) are generated on RS/1 (BBN Software, Boston, Mass.) run on aMicrovax computer.

EXAMPLE 10 Activation of Signaling Molecules in Human Cells

[0231] The following describes tests and results that are carried outevaluate the induction of some of the same signaling molecules involvedin stress responses as are activated by IL-1 alpha, IL-1 beta and otherinflammatory cytokines.

[0232] Human IL-1 eta is transfected into COS-1 cells. Several daysafter the transfection, conditioned medium (containing the transientlyexpressed IL-1 eta) is harvested. Test cells are incubated with thisconditioned medium, or alternatively with conditioned medium from COS-1cells transfected with the empty expression vector. Approximately 10minutes following the incubation, cell extracts are prepared from thetest cells, and the presence of activated signaling molecules is assayedby the use of antibodies specific for the phosphorylated forms ofIKBalpha (phosphorylation on Ser32), p38 MAP kinase (phosphorylation onThr180 and Tyr182), and Stress-Activated Protein Kinase (SAPK/JNK)(phosphorylation on Thr183/Tyr185). The antibodies may be obtained fromcommercial sources, such as New England Biolabs, Beverly, Mass. Thesesignal transduction molecules are known to be involved in a wide rangeof cellular responses to stimuli such as UV irradiation, endotoxin, andinflammatory cytokines including IL-1 beta. phosphorylation of one ormore of these molecules indicates that IL-1 eta is involved in stressresponse signaling pathways.

EXAMPLE 11 Activation of Cell Surface Molecules in Human Cells

[0233] The following describes tests that are carried out to evaluatethe ability of IL-1 eta to induce cell surface molecules involved instress responses (such as those that are induced by IL-1 alpha, IL-1beta and other inflammatory cytokines).

[0234] Human IL-1 eta is transfected into COS-1 cells. Several daysafter the transfection, conditioned medium (containing the transientlyexpressed IL-1 eta) is harvested. Human foreskin fibroblast (HFF) cellsare incubated for 18 hours at 37 degrees C. with this conditioned mediumdiluted 1:1 with fresh 0.5% serum-containing medium, or alternativelywith conditioned medium from control COS-1 cells transfected with theempty expression vector, diluted 1:1 with fresh 0.5% serum-containingmedium.

[0235] Following treatment with the conditioned medium from COS-1 cells,the HFF cells are washed twice with PBS and removed from the tissueculture vessel with versene (non-trypsin reagent). Cell-surface ICAM-1levels are measured by staining with anti-CD54-PE antibody (Pharmingen,San Diego, Calif.) on ice for one hour followed by washing andFACS-based detection. An increase in the level of cell-surface ICAM-1indicates that IL-1 eta is involved in upregulating cell-surfacemolecules that are induced during stress response.

EXAMPLE 12 Modulation of Cytokine Levels by IL-1 eta

[0236] The following describes tests that are carried out to evaluateinduction of cytokine secretion in dendritic cells or other cellscapable of secreting cytokines.

[0237] Monocyte-derived dendritic cells (MoDC) are obtained essentiallyas described by Pickl et al. (J. Immunol. 157:3850, 1996). Briefly,highly purified CD14(bright) peripheral blood monocytic cells areobtained from peripheral blood using an AutoMACS cell sorting system andanti-CD 14 magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach,Germany). The monocytic cells are cultured in the presence of IL-4 andGM-CSF for seven days to yield MoDC. Similar techniques are used toobtained purified or enriched populations of other cytokine-secretingcells, for example lymphocytes or granulocytes

[0238] Cells are treated for two to three days in the presence orabsence of IL-1 eta at varying concentrations; lipopolysaccharide (LPS)at 10 ng/ml is used as a positive control; heat-inactivated IL-1 eta(heated at 100 degrees C. for 30 minutes) may be used as a negativecontrol. Cells are separated from the supernatant medium bycentrifugation.

[0239] The supernatant medium is analyzed for soluble cytokine levelsusing a suitable assay (for example, the Luminex® multi-plex cytokineassay; Luminex Corporation, Austin, Tex.). Following culture, thesupernatant is harvested and assayed for several cytokines includingIL-10, IL-2, IL-4, IL-6, IL-8, IL-12 (p70 heterodimer), TNF-alpha,IFN-gamma, and GM-CSF.

[0240] For analysis of the induction of cytokine mRNA, the cells areharvested and total RNA is isolated (for example, using an RNeasy® TotalRNA System mini-kit, QIAGEN, Venlo, The Netherlands) and analyzed in asuitable, real-time quantitative polymerase chain reaction (PCR)analysis. Quantitative RT-PCR is performed using the ABI PRISM® 7700Sequence Detection System (Applied Biosystems, Foster City, Calif.) andTaqMan® reagents (Applied Biosystems). An increase in the levels of oneor more cytokines and/or induction of one or more cytokine mRNAsindicates that IL-1 eta upregulates cytokines that are involved in theinflammatory and/or immune response.

EXAMPLE 13 Effect of IL-1 eta on Mixed Lvmphocyte Reaction (MLR)

[0241] The following describes tests carried out to evaluate the effectsof IL-1 eta on TNF-alpha, IFN-gamma, and IL-10 secretion in a mixedleukocyte reaction (MLR) assay.

[0242] Briefly, highly purified CD14(bright) peripheral blood monocyticcells are obtained from peripheral blood using an AutoMACS cell sortingsystem and anti-CD 14 magnetic microbeads (Miltenyi Biotec, BergischGladbach, Germany). The monocytic cells are cultured in the presence ofIL-4 and GM-CSF for seven days to yield MoDC. Purified CD3+ allogeneic Tcells are obtained from freshly drawn blood using an AutoMACS cellsorting and anti-CD3 magnetic microbeads system (Miltenyi Biotec).

[0243] The allogeneic T cells are then mixed with MoDCs at a 1:10 MoDC:Tratio in quadruplicate in the presence of IL-1 eta at varyingconcentrations from 5 ng/ml to 200 ng/ml, or control preparations. Theensuing mixed lymphocyte reaction (MLR) is allowed to proceed for fourdays, and supernatants are harvested and assayed for TNF-alpha,IFN-gamma, and IL-10 using a suitable assay as described previously (forexample, the Luminex® multi-plex cytokine assay, DELFIA® or ELISAsubstantially as described below).

EXAMPLE 14 Cytokine ELISA

[0244] The following describes an Enzyme-Linked Immunosorbent Assay(ELISA) that is useful to detect and/or quantitate secreted proteins.The Example describes an assay specific for IL-10; those of skill in theart will recognize that a similar assay could be used to detect othermolecules.

[0245] ELISA plates (for example, Costar® EIA/RIA 96 well easy washplates, Corning Incorporated Life Sciences, Acton, Mass.) are coatedovernight with 100 microliter of a 2 micrograms/ml mixture ofRat-anti-huIL-10 capture antibody (BD Pharmingen, San Diego, Calif.) inbinding solution (0.1M NaH₂PO₄, pH 9.0) at 4 degrees C. Plates arewashed with wash buffer (phosphate buffered saline, or PBS, 0.5% Tween20) four times (400 microliters/well/wash), then one time with PBSwithout Tween. Plates were blocked with 100 microliters of 5% non-fatdry milk in PBS for 1 hour at room temperature (RT), and then washedwith wash buffer six times.

[0246] Samples and controls are added to separate wells (100microliters/well); serial dilutions of a standard protein, recombinantHuIL-10 (BD Pharmingen) in PBS+3% BSA (starting at 10 ng/ml in 3-folddilutions through 7 points as a standard curve, with an eighth point asa blank) is used to generate a standard curve for quantitation. Theplates are incubated for one hour at RT, then washed with wash buffersix times as previously described, and incubated withbiotinylated-rat-anti-HuIL-10 (BD Pharmingen; 100 microliters/well of a200 ng/ml mixture in PBS+3% BSA) for one hour at RT. The plates are thenwashed six times with wash buffer as before, and streptavidin-conjugatedhorse radish peroxidase (SA-HRP; Zymed Laboratories, Inc., South SanFrancisco, Calif.; 100 microliters/well of a 1:4000 dilution in PBS+3%BSA) is added.

[0247] After incubating at RT for 30 minutes, the plates are washed forthe final time as described above, and color is developed by adding 100microliters/well of Tetramethylbenzidene (TMB) substrate (a 1:1 mixtureof TMB Peroxidase Substrate: Peroxidase Solution, Kirkegaard & PerryLaboratories, Inc., Gaithersburg, Md.). The plates are incubated for 30minutes at RT, at which time color development is stopped with 100microliters/well of 2N H₂SO₄. The plates are read at 450 nm wavelengthon a Molecular Dynamics (Molecular Dynamics, Sunnyvale, Calif.) platereader, a standard curve is prepared, and the quantity of IL-10 in thesamples determined by comparison to the standard curve.

EXAMPLE 15 Cytokine DELFIA

[0248] The following describes a DELFIA® (dissociated enhancedlanthanide fluoroimmunoassay; PerkinElmer LifeSciences, Wallac Oy.,Turku, Finland) that is useful to detect and/or quantitate secretedproteins. The Example describes an assay specific for IL-10; those ofskill in the art will recognize that a similar assay could be used todetect other molecules.

[0249] Briefly, DELFIA® plates (i.e., Costar® high binding 96-wellplates, Corning Incorporated Life Sciences, Acton, Mass.) are coatedwith a detection (or capture) antibody (preferably a monoclonalantibody; 50 microliters of antibody solution containing 2 microgramsantibody/ml in PBS) at 4 degrees C. for 24 hours. Plates are washed withwash buffer (phosphate buffered saline, or PBS, 0.05% Tween 20) fourtimes (300 microliters/well/wash), then used in an assay or stored.

[0250] Fifty microliters each of test supernatants and cell specificcontrols are added to separate wells of an antibody-coated plate;dilutions of standard proteins are used to generate a standard curve forquantitation. Test supernatants and controls are incubated in theantibody coated plate to allow binding of cytokine to the antibody.Plates are then washed and a polyclonal biotinylated detection antibodyis added at a concentration of 10 nM in 50 microliters and incubated toallow binding to the captured cytokine. Plates are washed andStreptavidin-Europium (Eu) is added to the plate at a finalconcentration of 1 nM (0.06 micrograms/ml) in 50 microliters andincubated to allow binding to the biotinylated detection antibody.Plates are again washed and 100 microliters of enhancement solution isadded to bind the Eu. The Eu in solution is then detected by timeresolved fluorescence and the amount of cytokine secreted can bequantitated relative to standards which are added to each plate.

[0251] DELFIA® is amenable to full or partial automation (for example,using a Sagian Bioassay core system, Beckman Coulter, Inc., Fullerton,Calif., in combination with a plate reader such as a VICTOR2™,PerkinElmer LifeSciences), thereby rendering it useful forhigh-throughput testing.

EXAMPLE 16 Mouse Inflammatory Bowel Disease Models

[0252] This example describes several mouse models of inflammatory boweldisease (IBD), which includes Crohn's Disease and ulcerative colitis.Inflammatory bowel disease in animals can either occur spontaneously orcan be experimentally induced. It is necessary to exercise care whenselecting IBD models to study to ensure that the particular modelselected appropriately represents the relevant stage of the inflammatoryprocess under investigation. Particularly useful models of IBD include:

[0253] A. Oral Administration of Dextran Sulfate Sodium (DSS)

[0254] The DSS induction model can be used to induce either chronic oracute IBD. In the acute protocol, mice are given DSS (preferably with amolecular weight of 40 Kd; from 2% to 8%) in their drinking water forfrom one to eight days. The percent DSS and the duration of inductionwill vary depending on the strain of mouse used (for example, C3H/HeJ,C3H/He/Bir, NOD and NOD/SCID mice are highly susceptible, DBA/2,C57BL/6. BALB/c and 129/SvJ mice are moderately susceptible, withvarying degrees of susceptibility relative to each other, FVB mice aremoderately resistant, and NON/Ltj mice are resistant to DSS inducedcolitis). In the acute model, DSS is withdrawn after the inductionphase. To induce chronic colitis, 2-8% DSS is administered for from 5 toseven days followed by administration of water for ten days; this cycleis repeated three to four times.

[0255] DSS-induced colitis is marked by profound inflammation in thecolon of animals characterized by crypt destruction, mucosal ulceration,erosions and infiltration of lymphocytes and neutrophils into themucosal tissue. Histopathologic changes are individually scored as 0 (nofindings), 1 (minimal), 2 (mild), 3 (moderate), 4 (severe) for each ofthe following parameters: increased lymphocytes, increased neutrophils,ulceration, edema, crypt degeneration, and crypt regeneration. Totallesion score, crypt length and number of ulcers are also determined andused to gage severity of colitis.

[0256] B. Anti-CD3-Induced Ileitis

[0257] Mice (for example, BALB/c, C57BL/6 or MPJ mice, 6-16 weeks ofage) are given a single intraperitoneal (i.p.) injection ofanti-CD3epsilon antibody or control Ab (50 micrograms diluted in 500microliters PBS, pH 7.4). In wildtype mice such as those listed above,this treatment reliably induces diarrhea without being lethal.Immunosuppressants such as cyclosporin A (CsA, 50 mg/kg) ordexamethasone (Dex, 50 mg/kg) may be given i.p. either as a single doseat the same time as anti-CD3 antibody, or daily for a total of threeinjections beginning at the time of anti-CD3 injection, as controlmolecules that downregulate any ensuing immune response and prevent orameliorate anti-CD3-induced ileitis.

[0258] Mice are monitored for clinical signs of ileitis; mice may besacrificed at varying time points for histopathologic analysis and/ortesting by other means to evaluate apoptosis in gut tissue. Forhistopathology, hematoxylin and eosin (H&E) stained tissue sections ofparaffin embedded intestinal specimens are graded in a blinded fashion,for example by using a quantitative histology score based on thefrequency of apoptotic epithelial cells within the epithelium and theratio of villus height to crypt length. Histological alterations of thesmall intestinal mucosa that may be observed include a reduced villusheight, increased thickness of the crypt region, loss of Paneth cells,goblet cells and IEL in the epithelial layer and severe morphologicchanges of the epithelial cells. In the villi, the enterocytes may havelost their columnar and polarized morphology and become flattened. Inthe crypt region, numerous apoptotic bodies may identified in theepithelium.

[0259] C. MdrKO Spontaneous Colitis

[0260] The MDR gene family was identified by an ability to confermultiple drug resistance in cell lines. Three genes have been identifiedin rodents (mdr1, mdr2 and mdr3), and two in humans (MDR1, MDR3). Themouse mdr1a gene encodes a 170 kDa transmembrane protein that isexpressed in many tissues, including intestinal epithelial cells andsubsets of lymphoid and hematopoietic cells. Its function in these cellsis currently unknown, however, mice deficient in mdr1a spontaneouslydevelop colitis. In humans, MDR1 may be associated with IBDsusceptibility (Satsangi et al., Nat. Genet. 14:199, 1996; Brant et al.,Gastroenterology, 118:A331, 2000), while decreased MDR1 expression hasbeen reported in mucosal tissue from both CD and UC patients (Lawranceet al., Hum. Mol. Genet. 10: 445, 2001; Farrell et al.,Gastroenterology, 118:279, 2000). Mdr1a knockout mice (MdrKO) provide amodel of both acute (spontaneous) and chronic (DSS-induced) IBD, similarto that seen in humans, where IBD is generally a mixture of both chronicand acute inflammation. Acute colitis in MdrKO mice is marked by thespontaneous appearance of diarrhea and bloody stools in a subset of themice; chronic colitis is induced by administering 3% w/v DSS for sevendays in drinking water, followed by normal water.

[0261] Histopathologic changes are individually scored as 0 (nofindings), 1 (minimal), 2 (mild), 3 (moderate), 4 (severe) for each ofthe following parameters: increased mononuclear cells, increasedneutrophils, ulceration, edema, crypt degeneration, and hyperplasia.

[0262] D. Helicobacter-Induced Colitis

[0263] Various strains of mice with immunologic defects (i.e.,IL-10^(−/−) mice, recombinase-activating gene (Rag)1^(−/−) mice, T-cellreceptor alpha (TCRalpha)^(−/−) mice) are susceptible to colitis inducedby infection with Helicobacter spp., as described in Burich et al. (Am JPhysiol Gastrointest Liver Physiol 281:G764, 2001). Moreover, luminalbacteria appear to be an important factor contributing to thedevelopment of IBD in mice and humans. Accordingly, introduction ofHelicobacter spp. into immunodeficient mice also serves as an animalmodel of IBD humans (Burich et al. supra). In MdrKO mice, differentspecies of Helicobacter may have different effects on spontaneouscolitis; H. bilis infection induces IBD at a much earlier age, and thephenotypic appearance of Helicobacter-induced disease is similar, butnot identical, to spontaneous IBD. In contrast, there is minimal diseasein H. hepaticus-infected mr1a−/− mice, and H. hepaticus appears to delayonset of spontaneous IBD. Accordingly, those of skill in the art canutilize a Helicobacter-based model of IBD substantially as described byBurich et al. supra.

EXAMPLE 17 Mouse Asthma Models

[0264] This example describes a mouse model of asthma. Mice (forexample, BALB/c) are sensitized with antigen (for example, ovalbumin[OVA]) by intraperitoneal injection of the antigen in alum. Severalsensitization schemes are known in the art; a preferred scheme is toinject 10 micrograms of OVA three times at one week intervals (i.e., onday −21, day −14 and day −7). The mice are then challenged with antigeneither by aerosol exposure (5% OVA) or intranasal administration (0.1 mgOVA). The challenge schedule may be selected from among shorter terms(i.e., daily challenge on days 1, 2 and 3) or longer terms (i.e., weeklychallenge for two to three weeks). The endpoints that are measured caninclude airway hyperreactivity, bronchoalveolar lavage (BAL) cell numberand composition, in vitro draining lung lymph node cytokine levels,serum IgE levels, and histopathologic evaluation of lung tissue. Otheranimal models of asthma are known, and include the use of other animals(for example, C57BL/6 mice), sensitization schemes (for example,intranasal inoculation, use of other adjuvants or no adjuvants, etc.)and/or antigens (including peptides such as those derived from OVA orother proteinaceous antigens, ragweed extracts or other extracts such asthose used in desensitization regimens, etc.).

EXAMPLE 18 Mouse Collagen Induced Arthritis Model

[0265] This example describes two mouse models of rheumatoid arthritis,both of which are induced by immunization with collagen (eg.,collagen-induced arthritis or CIA). One model is dependant on tumornecrosis factor (TNF), the other is TNF-independent. Those of skill inthe art recognize that other animals models of rheumatoid arthritisexist, and further that various parameters within the models can beadjusted (see, for example, Luross and Williams, Immunology 103:407,2001; Schaller et al., Nat Immunol 2:74, 2001; Bober et al., ArthritisRheum 43:2660, 2000; or Weyand, C. M. in Rheumatology (Oxford) 2000June, pgs:3-8)).

[0266] TNF-dependent CIA is induced in male, wild-type (wt) DBA/1 micesubstantially as a modification of the protocol described by Courtenay,J. S. et al. (Nature 283:666, 1980) by immunization of mice with Type IIcollagen (CII; 100-200 micrograms) in complete Freund's adjuvant (CFA),followed by a booster of CII (200 micrograms) in incomplete Freund'sadjuvant (IFA) approximately three weeks later. In untreated mice, CIAmanifests in the paws, with increasing severity over time.

[0267] TNF-independent CIA is induced in male TNF Receptor doubleknockout (TNFR DKO) mice substantially as described above. TNFR DKO miceare mice that lack functional TNF receptors (both p55 and p75), and aredescribed in Peschon, et al. (J. Immunol. 160:943, 1998). Briefly, micelacking functional p55 and p75 genes were generated in C57BL/6background by gene targeting in embryonic stem cells. The TNFR DKOC57BL/6 mice were back-crossed on to the DBA/1 genetic background toyield mice that were homozygous for H-2q and were susceptible todevelopment of CIA.

[0268] The severity of disease is judged by swelling and joint functionof each paw, using a score from 0 to 4 (0=normal, no swelling;1=swelling in 1 to 3 digits; 2=mild swelling in ankles, forepaws or morethan three digits; 3=moderate swelling in multiple joints; 4=severeswelling with loss of function). The score for each paw is totaled for acumulative score for each mouse; cumulative scores are totaled for themice in each experimental group to yield a mean clinical score.

EXAMPLE 19 Mouse Experimental Allergic Encephalomyelitis Model

[0269] This example describes two mouse models of demyelinatingconditions; experimental autoimmune encephalomyelitis (or EAE) isdesigned to duplicate the secondary, immune mediated demyelination thatoccurs in multiple sclerosis.

[0270] A. Myelin Oligodendrocyte Glycoprotein (MOG)-Induced EAE inC57BL/6 Mice

[0271] EAE is induced in female C57BL/6 mice substantially as describedby Mendel et al. (Eur. J. Immunol. 25:1951-59, 1995) by immunization ofmice with an antigen derived from rat myelin oligodendrocyteglycoprotein (preferably the MOG35-55 peptide described by Mendel etal., supra). Other encephalitogenic antigens may be used, including, forexample, whole spinal chord homogenate, purified whole myelin, myelinbasic protein, proteolipid protein, myelin associated glycoprotein,myelin-associated oligodendrocyte basic protein, or encephalitogenicpeptides derived from these antigens. The disease induction protocol ofMendel et al. may be modified to include the use of a lower dose ofMOG35-55 for immunization (see below), no booster immunization, and theuse of RIBI® adjuvant (Corixa Corporation, Seattle Wash.) instead ofcomplete Freund's adjuvant.

[0272] To induce EAE, groups of age and weight-matched mice are given adose of 100 micrograms of rat MOG35-55 emulsified in 0.2 ml RIBI®adjuvant and injected subcutaneously (for example, at three sitesdistributed over the shaved flank of a mouse). To induce EAE withaccelerated onset, mice may be given an intravenous injection 500 ngpertussis toxin (List Biological Laboratory Inc, Campbell, Calif.),administered 48 hours after administration of MOG35-55.

[0273] B. Proteolipid Protein (PLP)-Induced EAE in SJL Mice

[0274] The PLP/SJL model results in a relapsing-remitting course ofdisease that mimics the course often seen in MS; however, SJL mice aresusceptible to anaphylaxis, and care must be given in choosing andadministering therapeutic agents to avoid induction of an anaphylacticresponse. EAE is induced in female SJL mice substantially as describedby McRae et al. et al. (J. Neuroimmunol. 38:229, 1992) by immunizationof mice with an antigen derived from rat proteolipid protein (preferablythe PLP13-151(S) peptide described by McRae et al., supra). Otherencephalitogenic antigens may be used, including, for example, wholespinal chord homogenate, purified whole myelin, myelin basic protein,proteolipid protein, myelin associated glycoprotein myelin-associatedoligodendrocyte basic protein, or encephalitogenic peptides derived fromthese antigens. The disease induction protocol of McRae et al. may bemodified as described above. EAE is reliably induced in SJL/J miceactively immunized with PLP13-151(S) or another, suitable PLP-relatedantigen. Alternatively, EAE can be induced by adoptive transfer ofPLP-specific T cells.

[0275] Administration of FIL1 antagonist(s) or control for either orboth models is initiated on the day after administration of theencephalitogenic peptide (day 1) and continued through day 11. Varyinginjection schedules can be used to evaluate the efficacy of the FIL1antagonist(s). Each mouse is injected intraperitoneally every other day(or according to the selected injection schedule) with 0.2 mlpyrogen-free phosphate-buffered saline (PBS) or 0.2 ml PBS containingFIL1 antagonist(s) or control. Endotoxin levels are monitored and mustbe less that <10 EU/mg of protein for all reagents. Mice are monitoreddaily for 30 to 35 days for weight loss, disease onset and severity ofclinical signs of EAE by an independent observer blinded to thetreatment groups.

[0276] The severity of EAE is assessed using either a standard EAE indexsystem in which “0” is used to indicate an asymptomatic mouse andclinical scores ranging from 0.5 to 4 are used to indicate varyingdegrees of ascending paralysis, or a slightly modified version of thecommonly used EAE scoring system. In the latter system, “0” indicates amouse with no evidence of disease and scores of 1-5 indicate varyingdegrees of ascending paralysis as follows: 1, tail paralysis; 2, hindlimb weakness; 3, partial hind limb paralysis; 4, complete hind limbparalysis; 5, moribund or dead. The disease protocol described aboveinduces an acute episode of disease in control mice (peak score of 24)from which most recover at least partially. Thus the acute episode ofdisease is not lethal and mice do not reach a score of 5. Theaforedescribed scale may be modified to include a score of “0.5” whichis given to mice that show the earliest signs of EAE but that do notexhibit complete paralysis of the tail. Mice given a score of 0.5exhibit some or all of the following symptoms: overnight weight loss of1-2 grams; noticeable tremor when held up by the tail; and weakness atthe distal tip of the tail.

[0277] The median day of onset of EAE is determined by Kaplan-MeierSurvival analysis. Significant differences in onset between groups areassessed using a Log-Rank comparison. Fischer's exact test is used toanalyze the statistical significance of differences in the incidence ofEAE among the groups of mice.

EXAMPLE 20 Mouse Cuprizone-Induced Demyelinating Disease Model

[0278] This example describes a mouse model (cuprizone-induceddemyelinating disease or CIDD) that is designed to mimic a type ofdemyelination that occurs in some cases of multiple sclerosis referredto as primary demyelination. CIDD is induced by feeding cuprizone(bis-cyclohexanone-oxaldihydrazone, a copper chelator) to micesubstantially as described by Matsushima et al. (Brain Pathol. 11:107,2001). At low doses of cuprizone, mature oligodendrocytes in the CNS arespecifically insulted and they become unable to provide support formyelin. Demyelination occurs when the damaged myelin is stripped fromthe axons by microglia.

[0279] Some advantages of the CIDD model are that it reproduciblyresults in massive demyelination in a large area of the mouse brain andit is reversible if cuprizone is removed from the diet. The modelappears well suited for profiling gene expression during various stagesof demyelination and remyelination. The model has been established inC57BL/6 mice, so it is also suitable for use in KO (knockout) or Tg(transgenic) mice with the B6 background. However, there are no obviousclinical signs associated with the demyelinating process, so analysismust be done by histology.

1 9 1 585 DNA Homo sapiens CDS (112)..(585) 1 ggcacgaggt tcctccccactctgtctttc tcacctctcc ttcacttttc ctagcctcct 60 caccaccatc tgatctatcttgttctcttc acaaaaggct ctgaagacat c atg aac 117 Met Asn 1 cca caa cgg gaggca gca ccc aaa tcc tat gct att cgt gat tct cga 165 Pro Gln Arg Glu AlaAla Pro Lys Ser Tyr Ala Ile Arg Asp Ser Arg 5 10 15 cag atg gtg tgg gtcctg agt gga aat tct tta ata gca gct cct ctt 213 Gln Met Val Trp Val LeuSer Gly Asn Ser Leu Ile Ala Ala Pro Leu 20 25 30 agc cgc agc att aag cctgtc act ctt cat tta ata gcc tgt aga gac 261 Ser Arg Ser Ile Lys Pro ValThr Leu His Leu Ile Ala Cys Arg Asp 35 40 45 50 aca gaa ttc agt gac aaggaa aag ggt aat atg gtt tac ctg gga atc 309 Thr Glu Phe Ser Asp Lys GluLys Gly Asn Met Val Tyr Leu Gly Ile 55 60 65 aag gga aaa gat ctc tgt ctcttc tgt gca gaa att cag ggc aag cct 357 Lys Gly Lys Asp Leu Cys Leu PheCys Ala Glu Ile Gln Gly Lys Pro 70 75 80 act ttg cag ctt aag gaa aaa aatatc atg gac ctg tat gtg gag aag 405 Thr Leu Gln Leu Lys Glu Lys Asn IleMet Asp Leu Tyr Val Glu Lys 85 90 95 aaa gca cag aag ccc ttt ctc ttt ttccac aat aaa gaa ggc tcc act 453 Lys Ala Gln Lys Pro Phe Leu Phe Phe HisAsn Lys Glu Gly Ser Thr 100 105 110 tct gtc ttt cag tca gtc tct tac cctggc tgg ttc ata gcc acc tcc 501 Ser Val Phe Gln Ser Val Ser Tyr Pro GlyTrp Phe Ile Ala Thr Ser 115 120 125 130 acc aca tca gga cag ccc atc tttctc acc aag gag aga ggc ata act 549 Thr Thr Ser Gly Gln Pro Ile Phe LeuThr Lys Glu Arg Gly Ile Thr 135 140 145 aat aac act aac ttc tac tta gattct gtg gaa taa 585 Asn Asn Thr Asn Phe Tyr Leu Asp Ser Val Glu 150 1552 157 PRT Homo sapiens 2 Met Asn Pro Gln Arg Glu Ala Ala Pro Lys Ser TyrAla Ile Arg Asp 1 5 10 15 Ser Arg Gln Met Val Trp Val Leu Ser Gly AsnSer Leu Ile Ala Ala 20 25 30 Pro Leu Ser Arg Ser Ile Lys Pro Val Thr LeuHis Leu Ile Ala Cys 35 40 45 Arg Asp Thr Glu Phe Ser Asp Lys Glu Lys GlyAsn Met Val Tyr Leu 50 55 60 Gly Ile Lys Gly Lys Asp Leu Cys Leu Phe CysAla Glu Ile Gln Gly 65 70 75 80 Lys Pro Thr Leu Gln Leu Lys Glu Lys AsnIle Met Asp Leu Tyr Val 85 90 95 Glu Lys Lys Ala Gln Lys Pro Phe Leu PhePhe His Asn Lys Glu Gly 100 105 110 Ser Thr Ser Val Phe Gln Ser Val SerTyr Pro Gly Trp Phe Ile Ala 115 120 125 Thr Ser Thr Thr Ser Gly Gln ProIle Phe Leu Thr Lys Glu Arg Gly 130 135 140 Ile Thr Asn Asn Thr Asn PheTyr Leu Asp Ser Val Glu 145 150 155 3 8 PRT Artificial FLAG 3 Asp TyrLys Asp Asp Asp Asp Lys 1 5 4 27 PRT artificial sequence lung SPDleucine zipper peptide 4 Pro Asp Val Ala Ser Leu Arg Gln Gln Val Glu AlaLeu Gln Gly Gln 1 5 10 15 Val Gln His Leu Gln Ala Ala Phe Ser Gln Tyr 2025 5 33 PRT artificial sequence leucine zipper 5 Arg Met Lys Gln Ile GluAsp Lys Ile Glu Glu Ile Leu Ser Lys Ile 1 5 10 15 Tyr His Ile Glu AsnGlu Ile Ala Arg Ile Lys Lys Leu Ile Gly Glu 20 25 30 Arg 6 30 DNA Homosapiens 6 acatcatgaa cccacaacgg gaggcagcac 30 7 28 DNA Homo sapiens 7ctctatcctg gaaccagcca cccacagc 28 8 30 DNA Homo sapiens 8 ccaaatcctatgctattcgt gattctcgac 30 9 29 DNA Homo sapiens 9 ggatttattc cacagaatctaagtagaag 29

1-23. (canceled)
 24. A polynucleotide comprising a polynucleotide thatis at least 98% identical to SEQ ID NO:1 and that encodes a polypeptidethat activates MAP kinase
 25. An expression vector comprising thepolynucleotide of claim
 24. 26. A host cell comprising the expressionvector of claim
 25. 27. A process for preparing a polypeptide comprisingculturing a host cell of claim 26 under conditions that promotesexpression of the polypeptide.